Renal effects of intrathecally injected tachykinins in the conscious saline-loaded rat: receptor and mechanism of action

1. The effects of intrathecally (i.t.) injected substance P (SP), neurokinin A (NKA), [β-Ala⁸]NKA (4–10) and [MePhe⁷]neurokinin B (NKB) at T₁₃ thoracic spinal cord level were investigated on renal excretion of water, sodium and potassium in the conscious saline-loaded rat. Antagonists selective for NK₁ (RP 67580), NK₂ (SR 48968) and NK₃ (R 820; 3-indolylcarbonyl-Hyp-Phg-N(Me)-Bzl) receptors were used to characterize the spinal effect of SP on renal function.
2. Saline gavage (4.5% of the body weight) enhanced renal excretion of water, sodium and potassium over the subsequent hour of measurement. Whereas these renal responses were not affected by 0.65 nmol SP, the dose of 6.5 nmol SP blocked the natriuretic response (aCSF value 3.9±0.8; SP value 0.7±0.3 μmol min⁻¹, P<0.01) as well as the renal excretion of water (aCSF value 48.9±5.8; SP value 14.5±4.0 μl min⁻¹, P<0.01) and potassium (aCSF value 4.8±0.6; SP value 1.5±0.6 μmol min⁻¹, P<0.01) at 30 min post-injection. SP had no significant effect on urinary osmolality. The SP-induced renal inhibitory effects during the first 30 min were abolished in rats subjected to bilateral renal denervation 1 week earlier or in rats injected i.t. 5 min earlier with 6.5 nmol RP 67580. In contrast, the co-injection of SR 48968 and R 820 (6.5 nmol each) did not affect the inhibitory responses to SP. On their own, these antagonists had no direct effect on renal excretion function. Since SP induced only transient changes in mean arterial blood pressure (−18.8±3.8 mmHg at 1 min and +6.3±2.4 mmHg at 5 min post-injection), it is unlikely that the renal effects of SP are due to systemic haemodynamic changes.
3. NKA (6.5 nmol but not 0.65 nmol) produced a transient drop in renal excretion of water (aCSF value 31.2±5.1; NKA value 11.3±4.2 μl min⁻¹, P<0.05), sodium (aCSF value 1.7±0.8; NKA value 0.4±0.2 μmol min⁻¹, P<0.05) and potassium (aCSF value 4.1±0.7; NKA value 1.5±0.4 μmol min⁻¹, P<0.05) at 15 min post-injection. However, the same doses (6.5 nmol) of selective agonists for tachykinin NK₂ ([β-Ala⁸]NKA(4-10)) and NK₃ ([MePhe⁷]NKB) receptors were devoid of renal effects.
4. This study provided functional evidence that tachykinins may be involved in the renal control of water and electrolyte excretion at the level of the rat spinal cord through the activation of NK₁ receptors and the sympathetic renal nerve.

     

Characterization of prejunctional 5-HT1 receptors that mediate the inhibition of pressor effects elicited by sympathetic stimulation in the pithed rat

1. A study was made of the effects of 5-carboxamidotryptamine (5-CT) on pressor responses induced in vivo by electrical stimulation of the sympathetic outflow from the spinal cord of pithed rats. All animals had been pretreated with atropine. Sympathetic stimulation (0.1, 0.5, 1 and 5 Hz) resulted in frequency-dependent increases in blood pressure. Intravenous infusion of 5-CT at doses of 0.01, 0.1 and 1 μg kg⁻¹ min⁻¹ reduced the pressor effects obtained by electrical stimulation. The inhibitory effect of 5-CT was significantly more pronounced at lower frequencies of stimulation. In the present study we characterized the pharmacological profile of the receptors mediating the above inhibitory effect of 5-CT.
2. The inhibition induced by 0.01 μg kg⁻¹ min⁻¹ of 5-CT on sympathetically-induced pressor responses was partially blocked after i.v. treatment with methiothepin (10  μg kg⁻¹), WAY-100,635 (100 μg kg⁻¹) or GR127935T (250 μg kg⁻¹), but was not affected by cyanopindolol (100 μg kg⁻¹).
3. The selective 5-HT_1A receptor agonist 8-OH-DPAT and the selective 5-HT_1B/1D receptor agonists sumatriptan and L-694,247 inhibited the pressor response, whereas the 5-HT_1B receptor agonists CGS-12066B and CP-93,129 and the 5-HT_2C receptor agonist m-CPP did not modify the pressor symapthetic responses.
4. The selective 5-HT_1A receptor antagonist WAY-100,635 (100 μg kg⁻¹) blocked the inhibition induced by 8-OH-DPAT and the selective 5-HT_1B/1D receptor antagonist GR127935T (250 μg kg⁻¹) abolished the inhibition induced either by L-694,247 or sumatriptan.
5. None of the 5-HT receptor agonists used in our experiments modified the pressor responses induced by exogenous noradrenaline (NA).
6. These results suggest that the presynaptic inhibitory action of 5-CT on the electrically-induced pressor response is mediated by both r-5-HT_1D and 5-HT_1A receptors.

     

Facilitation and inhibition of male rat ejaculatory behaviour by the respective 5-HT1A and 5-HT1B receptor agonists 8-OH-DPAT and anpirtoline,as evidenced by use of the corresponding new and selective receptor antagonists NAD-299 and NAS-181

1. Ejaculatory problems and anorgasmia are well-known side-effects of the SSRI antidepressants, and a pharmacologically induced increase in serotonergic neurotransmission inhibits ejaculatory behaviour in the rat. In the present study the role of 5-HT_1A and 5-HT_1B receptors in the mediation of male rat ejaculatory behaviour was examined by use of selective agonists and antagonists acting at these 5-HT receptor subtypes.
2. The 5-HT_1A receptor agonist 8-OH-DPAT (0.25–4.00 μmol kg⁻¹ s.c.) produced an expected facilitation of the male rat ejaculatory behaviour, and this effect was fully antagonized by pretreatment with the new selective 5-HT_1A receptor antagonist (R)-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide hydrogen (2R,3R) tartrate monohydrate (NAD-299) (1.0 μmol kg⁻¹ s.c.). NAD-299 by itself (0.75–3.00 μmol kg⁻¹ s.c.) did not affect the male rat ejaculatory behaviour.
3. The 5-HT_1B receptor agonist anpirtoline (0.25–4.00 μmol kg⁻¹ s.c.) produced a dose-dependent inhibition of the male rat ejaculatory behaviour, and this effect was fully antagonized by pretreatment with the 5-HT_1B receptor antagonist isamoltane (16 μmol kg⁻¹ s.c.) as well as by the new and selective antagonist (R)-(+)-2-(3-morpholinomethyl-2H-chromene-8-yl)oxymethylmorpholino methansulphonate (NAS-181) (16 μmol kg⁻¹ s.c.). Isamoltane (1.0–16.0 μmol kg⁻¹ s.c.) and NAD-181 (1.0–16.0 μmol kg⁻¹ s.c.) had no, or weakly facilitatory effects on the male rat ejaculatory behaviour. The non-selective 5-HT₁ receptor antagonist (−)-pindolol (8 μmol kg⁻¹ s.c.), did not antagonize the inhibition produced by anpirtoline.
4. The present results demonstrate opposite effects, facilitation and inhibition, of male rat ejaculatory behaviour by stimulation of 5-HT_1A and 5-HT_1B receptors, respectively, suggesting that the SSRI-induced inhibition of male ejaculatory dysfunction is due to 5-HT_1B receptor stimulation.

     

Mediation of 5-HT-induced external carotid vasodilatation in GR 127935-pretreated vagosympathectomized dogs by the putative 5-HT7 receptor

1. The vasodilator effects of 5-hydroxytryptamine (5-HT) in the external carotid bed of anaesthetized dogs with intact sympathetic tone are mediated by prejunctional sympatho-inhibitory 5-HT_1B/1D receptors and postjunctional 5-HT receptors. The prejunctional vasodilator mechanism is abolished after vagosympathectomy which results in the reversal of the vasodilator effect to vasoconstriction. The blockade of this vasoconstrictor effect of 5-HT with the 5-HT_1B/1D receptor antagonist, GR 127935, unmasks a dose-dependent vasodilator effect of 5-HT, but not of sumatriptan. Therefore, the present study set out to analyse the pharmacological profile of this postjunctional vasodilator 5-HT receptor in the external carotid bed of vagosympathectomized dogs pretreated with GR 127935 (20 μg kg⁻¹, i.v.).
2. One-minute intracarotid (i.c.) infusions of 5-HT (0.330 μg min⁻¹), 5-carboxamidotryptamine (5-CT; 0.010.3 μg min⁻¹), 5-methoxytryptamine (1100 μg min⁻¹) and lisuride (31000 μg min⁻¹) resulted in dose-dependent increases in external carotid blood flow (without changes in blood pressure or heart rate) with a rank order of agonist potency of 5-CT>>5-HT⩾5-methoxytryptamine>lisuride, whereas cisapride (1001000 μg min⁻¹, i.c.) was practically inactive. Interestingly, lisuride (mean dose of 85±7 μg kg⁻¹, i.c.), but not cisapride (mean dose of 67±7 μg kg⁻¹, i.c.), specifically abolished the responses induced by 5-HT, 5-CT and 5-methoxytryptamine, suggesting that a common site of action may be involved. In contrast, 1 min i.c. infusions of 8-OH-DPAT (33000 μg min⁻¹) produced dose-dependent decreases, not increases, in external carotid blood flow and failed to antagonize (mean dose of 200±33 μg kg⁻¹, i.c.) the agonist-induced vasodilator responses.
3. The external carotid vasodilator responses to 5-HT, 5-CT and 5-methoxytryptamine were not modified by intravenous (i.v.) pretreatment with either saline, (±)-pindolol (4 mg kg⁻¹) or ritanserin (100 μg kg⁻¹) plus granisetron (300 μg kg⁻¹), but were dose-dependently blocked by i.v. administration of methiothepin (10 and 30 μg kg⁻¹, given after ritanserin plus granisetron), mesulergine (10 and 30 μg kg⁻¹), metergoline (1 and 3 mg kg⁻¹), methysergide (1 and 3 mg kg⁻¹) or clozapine (0.3 and 1 mg kg⁻¹). Nevertheless, the blockade of the above responses, not significant after treatment with the lower of the two doses of metergoline and mesulergine, was nonspecific after administration of the higher of the two doses of methysergide and clozapine.
4. Based upon the above rank order of agonist potencies and the antagonism produced by a series of drugs showing high affinity for the cloned 5-ht₇ receptor, our results indicate that the 5-HT receptor mediating external carotid vasodilatation in GR 127935-pretreated vagosympathectomized dogs is operationally similar to the putative 5-HT₇ receptor mediating relaxation of vascular and non-vascular smooth muscles (e.g. rabbit femoral vein, canine coronary artery, rat systemic vasculature and guinea-pig ileum) as well as tachycardia in the cat.

     

Selectivity of action of 8-alkylamino analogues of N6-cyclopentyladenosine in vivo: haemodynamic versus anti-lipolytic responses in rats

1. A₁ adenosine receptor agonists with reduced intrinsic activity may be therapeutically useful as result of an increased selectivity of action. In this study the tissue selectivity of three 8-alkylamino substituted analogues of N⁶-cyclopentyladenosine (CPA) was investigated for haemodynamic and anti-lipolytic effects using an integrated pharmacokinetic-pharmacodynamic approach.
2. Chronically instrumented male Wistar rats received intravenous infusions of 4.0 mg kg⁻¹ 8-methylaminoCPA (8MCPA), 12.0 mg kg⁻¹ 8-ethylaminoCPA (8ECPA), 20.0 mg kg⁻¹ 8-butylaminoCPA (8BCPA) or vehicle during 15 min. During experimentation, serial arterial blood samples were drawn for the determination of agonist concentrations and plasma non-esterified fatty acid (NEFA) levels. Blood pressure and heart rate were monitored continuously. In addition to the CPA analogues, each rat received a rapid bolus infusion of CPA to determine the maximal effects of the full agonist.
3. The concentration-time profiles of the CPA analogues could be described by a bi-exponential function. Values for clearance, volume of distribution at steady state and elimination half-life were 44±5, 48±6 and 39±2 ml min⁻¹ kg⁻¹, 0.97±0.09, 0.84±0.10 and 1.05±0.07 1 kg⁻¹ and 25±2, 28±2 and 40±2 min for 8MCPA, 8ECPA and 8BCPA, respectively (mean±s.e.mean, n=6–8).
4. Different models were used to derive the concentration-effect relationships for heart rate and NEFA, yielding estimates of potency (EC₅₀) and instrinsic activity (E_max) for both effects of the compounds in vivo. On heart rate the compounds acted as partial agonists, with E_max values of −173±14, −131±11 and −71±6 beats min⁻¹ for 8MCPA, 8ECPA and 8BCPA, respectively. These E_max values were significantly lower than the maximal effect of CPA (−208±8 beats min⁻¹). With regard to the anti-lipolytic effect all three compounds were full agonists and lowered NEFA levels to the same extent as CPA (69%). The estimated E_max values were 63±5, 63±4 and 68±2%, respectively.
5. Furthermore, the compounds were more potent in causing anti-lipolytic than cardiovascular effects. The EC₅₀ values for the NEFA and heart rate lowering effects were 37±15, 68±22 and 659±108 ng ml⁻¹ and 164±22, 341±76 and 975±190 ng ml⁻¹ for 8MCPA, 8ECPA and 8BCPA, respectively (mean±s.e.mean, n=6–8).
6. This study demonstrates that partial agonists for the A₁ adenosine receptor have increased selectivity of action in vivo. The 8-alkylamino analogues of CPA may be useful anti-lipolytics with less pronounced haemodynamic side effects.

     

Dual effects of dichloroacetate on cardiac ischaemic preconditioning in the rat isolated perfused heart

1. Ischaemic cardiac preconditioning represents an important cardioprotective mechanism which limits myocardial ischaemic damage. The aim of this investigation was to assess the impact of dichloroacetate (DCA), a pyruvate dehydrogenase complex activator, on preconditioning.
2. Rat isolated hearts were perfused by use of the Langendorff technique, and were subjected to either preconditioning (3×4 or 3×6 min ischaemia) or continuous perfusion, followed by 30 min global ischaemia and 60 min reperfusion. DCA (3 mM) was either given throughout the protocol (pretreatment), during reperfusion only (post-treatment), or not at all. Throughout reperfusion mechanical performance was assessed as the rate-pressure product (RPP: left ventricular developed pressure×heart rate).
3. In non-preconditioned control hearts, mechanical performance was substantially (P<0.001) depressed on reperfusion (the RPP after 60 min of reperfusion (RPP_t=60) was 4,246±974 mmHg beats min⁻¹ compared to baseline value of 21,297±1,728 mmHg beats min⁻¹). Preconditioning with either 3×4 min or 3×6 min cycles caused significant protection, as shown by enhanced recovery (RPP_t=60=7,818±1,138, P<0.05, and 11,123±587 mmHg beats min⁻¹, P<0.001, respectively).
4. Addition of DCA (3 mM) to hearts under baseline conditions significantly (P<0.001) enhanced systolic function with an increased left ventricular developed pressure of 108±5 mmHg compared to 88.3±3.0 mmHg in the controls.
5. Pretreatment with 3 mM DCA had no effect on recovery of mechanical performance in the non-preconditioned hearts (RPP_t=60=3,640±1,235 mmHg beats min⁻¹) while the beneficial effects of preconditioning were reduced in the preconditioned hearts (3×4 min: RPP_t=60=2,919±1,060 mmHg beats min⁻¹; 3×6 min: RPP_t=60=8,032±1,367 mmHg beats min⁻¹). Therefore, DCA had increased the threshold for preconditioning.
6. By contrast, post-treatment of hearts with 3 mM DCA substantially improved recovery on reperfusion in all groups (RPP_t=60=5,827±1,328 (non-preconditioned), 14,022±3,743 (3×4 min; P<0.01) and 23,219±1,374 (3×6 min; P<0.001) mmHg beats min⁻¹).
7. The results of the present investigation clearly show that pretreatment with DCA enhances baseline cardiac mechanical performance but increases the threshold for cardiac preconditioning. However, post-treatment with DCA substantially augments the beneficial effects of preconditioning.

     

Endogenous angiotensin II and the reflex response to stimulation of cardiopulmonary serotonin 5HT3 receptors

1. Angiotensin (Ang) II modulates cardiovascular baroreflexes; whether or not the peptide influences chemosensitive cardiovascular reflexes is not known. We tested the hypothesis that Ang II modulates the reflex control of sympathetic nerve activity exerted by 5-hydroxytryptamine 3 (5HT₃) cardiopulmonary receptors.
2. The 5HT₃ receptor agonist phenylbiguanide (PBG), infused intravenously for 15 min, elicited a sustained reflex decrease of renal sympathetic nerve activity (RSNA) but only transient (<3 min) changes of arterial blood pressure (BP) and heart rate (HR) in methohexital-anaesthesized rats.
3. Infusion of Ang II at a dose that did not affect baseline BP, HR and RSNA enhanced the PBG-evoked reflex decrease of RSNA (−54±5% in Ang II treated versus −33±6% in control rats after 15 min PBG, P<0.05, n=6 each) in methohexital-anaesthetized rats.
4. The angiotensin converting enzyme (ACE) inhibitor lisinopril blunted the reflex responses to PBG in anaesthetized as well as conscious animals. The effect of the ACE inhibitor was abolished by concomitant infusion of Ang II.
5. The reflex response to stimulation of cardiopulmonary 5HT₃ afferents was also impaired by the Ang II type 1 receptor (AT₁) blocker ZD7155 but not by the type 2 (AT₂) blocker PD 123319.
6. Infusion of a volume load to stimulate cardiopulmonary baroreceptors induced a gradual decrease of RSNA which was impaired by exogenous Ang II (RSNA −26±6% in Ang II treated versus −47±6% in control rats after volume load, P<0.05, n=6 each) but unaffected by ACE inhibition.
7. The reflex control of RSNA by cardiopulmonary 5HT₃ receptors is enhanced by Ang II via AT₁ receptors. Thus, Ang II facilitates a chemosensitive cardiovascular reflex, in contrast to its inhibitory influences on mechanosensitive reflexes.

     

Improved metabolic status and insulin sensitivity in obese fatty (fa/fa) Zucker rats and Zucker Diabetic Fatty (ZDF) rats treated with the thiazolidinedione,MCC-555

1. We examined the effect of chronic (21 days) oral treatment with the thiazolidinedione, MCC-555 ((±)-5-[{6-(2-fluorbenzyl)-oxy-2-naphy}methyl]-2,4-thiazolidinedione) on metabolic status and insulin sensitivity in obese (fa/fa) Zucker rats and Zucker Diabetic Fatty (ZDF) rats which display an impaired glucose tolerance (IGT) or overt diabetic symptoms, respectively.
2. MCC-555 treatment to obese Zucker rats (10 and 30 mg kg⁻¹) and diabetic ZDF rats (10 mg kg⁻¹) reduced non-esterified fatty acid concentrations in both rat strains and reduced plasma glucose and triglyceride concentrations in the obese Zucker rats. Liver glycogen concentrations were significantly increased by chronic MCC-555 treatment in both obese Zucker rats (30 mg kg⁻¹ day⁻¹) and diabetic ZDF rats (10 mg kg⁻¹ day⁻¹), as compared with vehicle-treated lean and obese rats and there was a significant increase in hepatic glycogen synthase activity in MCC-555-treated diabetic ZDF rats as compared to vehicle-treated controls.
3. During a euglycaemic hyperinsulinaemic clamp, MCC-555-treated obese Zucker rats and diabetic ZDF rats required significantly higher glucose infusion rates to maintain stable glucose concentrations (2.01±0.19 mg min⁻¹ and 6.42±1.03 mg min⁻¹, respectively) than vehicle-treated obese controls (0.71±0.17 mg min⁻¹ and 2.09±0.71 mg min⁻¹; P<0.05), demonstrating improved insulin sensitivity in both Zucker and ZDF rats. MCC-555 treatment also enhanced insulin-induced suppression of hepatic glucose production in ZDF rats as measured using infusions of [6-³H]-glucose under clamp conditions.
4. In conclusion, we have demonstrated that MCC-555 improves metabolic status and insulin sensitivity in obese Zucker and diabetic ZDF rats. MCC-555 may prove a useful compound for alleviating the metabolic disturbances and IGT associated with insulin resistance in man.

     

Characterization of putative 5-HT7 receptors mediating tachycardia in the cat

1. It has been suggested that the tachycardic response to 5-hydroxytryptamine (5-HT) in the spinal-transected cat is mediated by ‘5-HT₁-like'' receptors since this effect, being mimicked by 5-carboxamidotryptamine (5-CT), is not modified by ketanserin or MDL 72222, but it is blocked by methiothepin, methysergide or mesulergine. The present study was set out to reanalyse this suggestion in terms of the IUPHAR 5-HT receptor classification schemes proposed in 1994 and 1996.
2. Intravenous (i.v.) bolus injections of the tryptamine derivatives, 5-CT (0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30 μg kg⁻¹), 5-HT (3, 10 and 30 μg kg⁻¹) and 5-methoxytryptamine (3, 10 and 30 μg kg⁻¹) as well as the atypical antipsychotic drug, clozapine (1000 and 3000 μg kg⁻¹) resulted in dose-dependent increases in heart rate, with a rank order of agonist potency of 5-CT >> 5-HT > 5-methoxytryptamine >> clozapine.
3. The tachycardic effects of 5-HT and 5-methoxytryptamine were dose-dependently antagonized by i.v. administration of lisuride (30 and 100 μg kg⁻¹), ergotamine (100 and 300 μg kg⁻¹) or mesulergine (100, 300 and 1000 μg kg⁻¹); the highest doses of these antagonists used also blocked the tachycardic effects of 5-CT. Clozapine (1000 and 3000 μg kg⁻¹) did not affect the 5-HT-induced tachycardia, but attenuated, with its highest dose, the responses to 5-methoxytryptamine and 5-CT. However, these doses of clozapine as well as the high doses of ergotamine (300 μg kg⁻¹) and mesulergine (300 and 1000 μg kg⁻¹) also attenuated the tachycardic effects of isoprenaline. In contrast, 5-HT-, 5-methoxytryptamine- and 5-CT-induced tachycardia were not significantly modified after i.v. administration of physiological saline (0.1 and 0.3 ml kg⁻¹), the 5-HT_1B/1D receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"GR127935","term_id":"238377770","term_text":"GR127935"}}GR127935 (500 μg kg⁻¹) or the 5-HT_3/4 receptor antagonist, tropisetron (3000 μg kg⁻¹).
4. Intravenous injections of the 5-HT₁ receptor agonists, sumatriptan (30, 100 and 300 μg kg⁻¹) and indorenate (300 and 1000 μg kg⁻¹) or the 5-HT₄ receptor (partial) agonist cisapride (300 and 1000 μg kg⁻¹) were devoid of effects on feline heart rate per se and failed to modify significantly 5-HT-induced tachycardic responses.
5. Based upon the above rank order of agonist potency, the failure of sumatriptan, indorenate or cisapride to produce cardioacceleration and the blockade by a series of drugs showing high affinity for the cloned 5-ht₇ receptor, the present results indicate that the 5-HT receptor mediating tachycardia in the cat is operationally similar to other putative 5-HT₇ receptors mediating vascular and non-vascular responses (e.g. relaxation of the rabbit femoral vein, canine external carotid and coronary arteries, rat systemic vasculature and guinea-pig ileum). Since these responses represent functional correlates of the 5-ht₇ gene product, the 5-HT₇ receptor appellation is reinforced. Therefore, the present experimental model, which is not complicated by the presence of other 5-HT receptors, can be utilized to characterize and develop new drugs with potential agonist and antagonist properties at functional 5-HT₇ receptors.

     

Effect of the selective 5-HT1A receptor antagonist WAY 100635 on the inhibition of e.p.s.ps produced by 5-HT in the CA1 region of rat hippocampal slices

1. The actions of N-(2-(-4(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexane carboxamide (WAY 100635), a novel and selective 5-hydroxytryptamine_1A (5-HT_1A) antagonist, on excitatory postsynaptic potentials (e.p.s.ps) were investigated by use of intracellular recordings in pyramidal cells of the CA1 region of rat hippocampal slices.
2. WAY 100635 (10 nM) did not affect any of the investigated parameters of cell excitability such as membrane potential, total input resistance (R_in), firing threshold, action potential amplitude, action potential frequency adaptation, and slow afterhyperpolarization (sAHP) which follows repetitive firing of action potentials. WAY 100635 did not have any effect on either the slope or the amplitude of e.p.s.ps evoked by stimulation of the CA1 stratum radiatum.
3. Bath application of either 5-hydroxytryptamine (5-HT, 10–30 μM) or 5-carboxamidotryptamine (5-CT, 300 nM) hyperpolarized the membrane potential (ΔV_m=−4.1±0.9 and −6.0±0.9 mV, respectively), and reduced R_in (−25±8% and −18±1%, respectively). 5-HT blocked the action potential frequency adaptation and significantly reduced the amplitude of the sAHP that follows repetitive firing of action potentials.
4. 5-HT significantly decreased the amplitude of evoked e.p.s.ps (−14±6%). This effect was greater in the presence of the GABA_A receptor antagonist bicuculline (10 μM, −45±12%) and was mimicked by 5-CT (−49±5%). Both AMPA and NMDA components of e.p.s.ps were significantly reduced in amplitude by 5–HT (−38±8%, n=6, and −29±12%, n=3, respectively; P<0.05).
5. WAY 100635 fully antagonized the hyperpolarization, the reduction of R_in, and the decrease in amplitude of e.p.s.ps elicited by 5-HT, while it did not affect the action of 5-HT on the action potential frequency adaptation. In the presence of WAY 100635, 5-HT elicited a depolarization which was blocked by 10–30 μM RS 23597-190, a selective 5-HT₄ receptor antagonist.
6. Our data demonstrate that WAY 100635 is devoid of direct effects on CA1 pyramidal cell excitability and on evoked e.p.s.ps, while it fully antagonizes the effects of 5-HT on excitatory synaptic transmission and on hyperpolarization, without affecting the 5-HT₄ receptor-mediated response. Since WAY 100635 selectively antagonizes 5-HT_1A receptor-mediated actions of 5-HT, our data also demonstrate that the inhibitory action of 5-HT on excitatory synaptic transmission in CA1 is mediated by 5-HT_1A receptors.

     

Systemic and renal effects of an ETA receptor subtype-specific antagonist in healthy subjects

1. Endothelins (ETs) might play a pathophysiological role in a variety of vascular diseases. The aim of the present study was to characterize the effects of BQ-123, a specific ET_A receptor antagonist on systemic and renal haemodynamics in healthy subjects. This was done at baseline and during infusion of exogenous ET-1.
2. The study was performed in a balanced, randomized, placebo-controlled, double blind 4 way cross-over design in 10 healthy male subjects. Subjects received co-infusions of ET-1 (2.5 ng kg⁻¹ min⁻¹ for 120 min) or placebo and BQ-123 (15 μg min⁻¹ for 60 min and subsequently 60 μg min⁻¹ for 60 min) or placebo. Renal plasma flow (RPF) and glomerular filtration rate (GFR) were assessed by the para-aminohippurate (PAH) and the inulin plasma clearance method, respectively.
3. BQ-123 alone had no renal or systemic haemodynamic effect. ET-1 significantly reduced RPF (−24%, P<0.001) and GFR (−12%, P=0.034). These effects were abolished by co-infusion of either dose of BQ-123 (RPF: P=0.0012; GFR: P=0.020).
4. BQ-123 reversed the renal haemodynamic effects induced by exogenous ET-1 in vivo. This indicates that vasoconstriction in the kidney provoked by ET-1 is predominantly mediated by the ET_A receptor subtype.

     

The effect of chronic treatment with trandolapril on cyclic AMP- and cyclic GMP-dependent relaxations in aortic segments of rats with chronic heart failure

1. Characteristics of cyclic GMP- and cyclic AMP-mediated relaxation in aortic segments of rats with chronic heart failure (CHF) and the effects of chronic treatment with an angiotensin I converting enzyme (ACE) inhibitor, trandolapril, were examined 8 weeks after coronary artery ligation.
2. Cardiac output indices of coronary artery-ligated and sham-operated rats were 125±8 and 189±10 ml min⁻¹ kg⁻¹, respectively (P<0.05), indicating the development of CHF at this period.
3. The maximal relaxant response of aortic segments to 10 μM acetylcholine in rats with CHF and sham-operated rats was 64.0±5.7 and 86.9±1.9%, respectively (P<0.05), whereas the relaxant response to sodium nitroprusside (SNP) remained unchanged. Tissue cyclic GMP content in rats with CHF was lower than that of sham-operated rats.
4. In endothelium-intact segments of rats with CHF, the maximal relaxant response to 10 μM isoprenaline (44.5±6.7%) was lower that sham-operated rats (81.3±2.5%, P<0.05) and the concentration-response curve for NKH477, a water-soluble forskolin, was shifted to the right without a reduction in the maximal response. Isoprenaline-induced relaxation of aortic segments was attenuated by N^G-nitro-L-arginine methyl ester (L-NAME) in sham-operated rats, but not in rats with CHF. Relaxation to 30 μM dibutyryl cyclic AMP in rats with CHF (26.8±2.7%) was lower than that in sham-operated rats (63.4±11.8%, P<0.05).
5. Trandolapril (3 mg kg⁻¹ day⁻¹) was orally administered from the 2nd to 8th week after the operation. Aortic blood flow of rats with CHF (38.5±3.6 ml min⁻¹) was lower than that of sham-operated rats (55.0±3.0 ml min⁻¹), and this reduction was reversed (54.1±3.4 ml min⁻¹) by treatment with trandolapril. The diminished responsiveness described above was normalized in the trandolapril-treated rat with CHF (i.e., the maximal relaxation to acetylcholine, 94.7±1.0%; that to isoprenaline, 80.5±2.8%; that to dibutyryl cyclic AMP, 54.7±6.2%). However, aortic segments of trandolapril-treated rats with CHF, L-NAME did not attenuate isoprenaline-induced relaxation and the tissue cyclic GMP level was not fully restored, suggesting that the ability of the endothelium to produce NO was still partially damaged.
6. The results suggest that vasorelaxation in CHF, diminished mainly due to dysfunction in endothelial nitric oxide (NO) production and cyclic AMP-mediated signal transduction, was partially restored by long-term treatment with trandolapril. The mechanism underlying the restoration may be attributed in part to prevention of CHF-induced endothelial dysfunction.

     

Effect of chronic bradykinin B2 receptor blockade on blood pressure of conscious Dahl salt-resistant rats

1. In this study 3 protocols were utilized to determine the role of endogenous kinins in the resistance of the inbred Dahl (Rapp) salt-resistant (SR/Jr) rats to high salt diet-induced blood pressure elevation.
2. The bradykinin B₂ receptor antagonist, Hoe 140 (D-Arg[Hyp³, Thi⁵, D-Tic⁷, Oic⁸]-bradykinin) at doses of either 10–20 or 20–40 nmol day⁻¹ (subcutaneously (s.c), via osmotic minipumps, for either 1 or 3 weeks during a high (8%) salt diet) effectively blocked or attenuated the hypotensive responses to 100–1000 ng of bradykinin.
3. In the first protocol, 5 week old SR/Jr rats treated with Hoe 140 (10–20 nmol day⁻¹, n=9, s.c., via osmotic minipumps) for 3 weeks and concomitantly fed high (8%) NaCl diet had significantly higher conscious tail cuff blood pressures (BPc) at 1 and 3 weeks when compared with rats treated with vehicle (0.9% NaCl, n=6). The differences in BPc between the 2 groups were 13 mmHg (P<0.001) after 1 week and 8 mmHg (P<0.05) after 3 weeks of treatment.
4. In the second protocol, 5 week old SR/Jr rats were treated with Hoe 140 (20–40 nmol day⁻¹, n=8, s.c., via osmotic minipumps) or vehicle (n=8) for 3 weeks. During the first week of treatment the rats were fed normal (0.8%) NaCl diet. The rats were then switched to 8% NaCl for 2 remaining weeks of the protocol. The mean BPc of Hoe 140-treated rats was not significantly different from that of the vehicle-treated rats when fed 0.8% NaCl diet. In contrast, rats treated with Hoe 140 and concomitantly fed high (8%) NaCl diet had significantly increased BPc (123±2 vs 111±1 mmHg, P<0.001 for the Hoe 140- and vehicle-treated rats, respectively).
5. In the third protocol, treatment with Hoe 140 (20–40 nmol day⁻¹, s.c., via osmotic minipumps) during high salt diet did not increase BPc in rats that were pre-exposed to the high salt diet for 2 weeks.
6. At the end of 3 weeks of study, blood pressure was measured via an arterial catheter during pentobarbitone-induced anaesthesia. Rats treated with Hoe 140 for 1 or 3 weeks had significantly lower mean arterial blood pressures than the vehicle-treated rats.
7. Our findings suggest that in SR/Jr rats, kinin activation of bradykinin B₂ receptors at least partially contributes to early regulatory mechanisms that resist an increase in blood pressure following exposure to a high salt diet. The mechanism underlying the decreased blood pressure during pentobarbitone anaesthesia of SR/Jr rats chronically treated with Hoe 140 has yet to be elucidated.

     

Effects of intracerebroventricular leptin administration on food intake,body weight gain and diencephalic nitric oxide synthase activity in the mouse

1. Intracranial administration of leptin reduces both food intake and body weight gain in the mouse. Inhibitors of nitric oxide (NO) synthase produce similar effects.
2. To investigate the role of the brain L-arginine/NO pathway in mediating this effect of leptin, we have evaluated food intake and body weight gain after daily (5 days) intracerebroventricular (i.c.v.) administration of leptin (0.5–2 μg) alone or in association with L-arginine (10 μg). Moreover, we measured diencephalic nitric oxide synthase (NOS) activity after a single i.c.v. leptin (0.25–2 μg) injection and after consecutive doses of leptin (0.25–2 μg) over 5 days. The time course of the effect of leptin on NOS activity was also evaluated.
3. I.c.v. injected leptin (1 and 2 μg) significantly and dose-dependently reduced food intake and body weight gain with respect to vehicle (food intake: 5.97±0.16 g 24 h⁻¹ and 4.27±0.18 g 24 h⁻¹, respectively, vs 8.05±0.34 g 24 h⁻¹, P<0.001, n=6 for each group; body weight gain: −10.7±0.46% and −15.2±0.65%, respectively, vs 5.14±0.38%, P<0.001, n=6 for each group). This effect was antagonized by L-arginine (food intake: 7.90±0.37 g 24 h; body weight gain: 5.11±0.31%, n=6). Diencephalic NOS activity was significantly reduced by the highest doses of leptin with respect to vehicle (vehicle: 0.90±0.04 nmol citrulline min⁻¹ g⁻¹ tissue; leptin 1 μg: 0.62±0.03 nmol citrulline min⁻¹ g⁻¹ tissue, P<0.001; leptin 2 μg: 0.44±0.03 nmol citrulline min⁻¹ g⁻¹ tissue, P<0.001, n=6 for each group). Similar results were obtained in animals treated with daily consecutive doses of leptin. The inhibitory effect appeared rapidly (within 30 min) and was long lasting (up to 12 h).
4. Our results suggest that the brain L-arginine/NO pathway may be involved in the central effect of leptin on feeding behaviour and body weight gain in mice.

     

Effects of tumour necrosis factor-α on the coronary circulation of the rat isolated perfused heart: a potential role for thromboxane A2 and sphingosine

1. The actions of tumour necrosis factor-α (TNF-α) on the coronary circulation were investigated in the rat isolated heart, perfused under constant flow, recirculating conditions.
2. An early increase in coronary perfusion pressure (CPP) was observed upon treatment with TNF-α (increase in CPP 10 min after TNF-α treatment: 45±12 mmHg vs control: 15±4 mmHg, P<0.05). The role of sphingosine, prostanoids and endothelins, in this coronary constrictor action, was investigated with the use of pharmacological inhibitors and antagonists.
3. The TNF-α induced increase in coronary tone was blocked by indomethacin, 10 μM (increase in CPP after 10 min: 13±4 mmHg vs TNF-α alone, P<0.05).
4. The thromboxane receptor antagonist GR32191, 10 μM, attenuated the TNF-α induced coronary constriction (12±2 mmHg vs TNF-α alone, P<0.05), as did the joint thromboxane A₂ synthesis inhibitor and receptor antagonist ZD1542, 10 μM (8±1 mmHg vs TNF-α alone, P<0.05).
5. The ceramidase inhibitor N-oleoylethanolamine (NOE), 1 μM, also blocked the TNF-α induced response (8±4 mmHg vs TNF-α alone, P<0.05).
6. In contrast, the coronary constrictor action of TNF-α was not inhibited by the endothelin_A/B receptor antagonist bosentan, 3 μM (38±9 mmHg vs TNF-α, P=NS).
7. These data indicated that the early coronary vasoconstriction induced by TNF-α was mediated by both thromboxane A₂ and sphingosine, suggesting an interaction between both the sphingomyelinase and phospholipase A₂ metabolic pathways.

     

Effect of binding protein surface charge on palmitate uptake by hepatocyte suspensions

1. Studies were directed at determining whether hepatocytes, isolated from female Sprague-Dawley rats, facilitate the uptake of protein-bound long-chain fatty acids. We postulated one form of facilitated uptake may occur through an ionic interaction between the protein-ligand complex and the cell surface. These interactions are expected to supply additional ligand to the cell for uptake.
2. The clearance rate of [³H]-palmitate in the presence of α₁-acid-glycoprotein (pI=2.7), albumin (pI=4.9) and lysozyme (pI=11.0) was investigated. Palmitate uptake was determined in the presence of protein concentrations that resulted in similar unbound ligand fractions (=0.03). The experimental clearance rates were compared to the theoretical predictions based upon the diffusion-reaction model.
3. By use of our experimentally determined equilibrium binding and dissociation rate constants for the various protein-palmitate complexes, the diffusion-reaction model predicted clearance rates were 4.9 μl s⁻¹/10⁶ cells, 4.8 μl s⁻¹/10⁶ cells and 5.5 μl s⁻¹/10⁶ cells for α₁-acid-glycoprotein, albumin and lysozyme, respectively; whereas the measured hepatocyte palmitate clearance rates were 1.2±0.1 μl s⁻¹/10⁶ cells, 2.3±0.3 μl s⁻¹/10⁶ cells and 7.1±0.7 μl s⁻¹/10⁶, respectively.
4. Hepatocyte palmitate clearance was significantly faster (P<0.01) in the presence of lysozyme than albumin which was significantly faster than α₁-acid-glycoprotein (P<0.01). The marked difference in clearance rates could not be explained by considering differences in solution viscosity.
5. Our results are consistent with the notion that ionic interactions between protein-ligand complexes and the cell surface facilitate the ligand uptake by decreasing the diffusional distance of the unbound ligand and/or by facilitating the protein-ligand dissociation rate.

     

Citalopram-induced hypophagia is enhanced by blockade of 5-HT1A receptors: role of 5-HT2C receptors

1. The selective 5-hydroxytryptamine reuptake inhibitor citalopram (10 and 20 mg kg⁻¹, i.p.) significantly reduced food intake in male rats (CD-COBS) habituated to eat their daily food during a 4-h period.
2. The 5-HT_1A receptor antagonist WAY100635 (0.3 mg kg⁻¹) administered systemically did not modify feeding but significantly potentiated the reduction in food intake caused by 10 mg kg⁻¹ i.p. citalopram. The dose of 5 mg kg⁻¹ i.p. citalopram was not active in animals pretreated with vehicle but significantly reduced feeding in animals pretreated with WAY100635.
3. WAY100635 (0.1 μg 0.5 μl⁻¹) injected into the dorsal raphe significantly potentiated the hypophagic effect of 10 mg kg⁻¹ citalopram.
4. WAY100635 (1.0 μg 0.5 μl⁻¹) injected into the median raphe did not modify feeding or the hypophagic effect of 10 mg kg⁻¹ citalopram.
5. The 5-HT_2B/2C receptor antagonist SB206553 (10 mg kg⁻¹, p.o.) slightly reduced feeding by itself but partially antagonized the effect of WAY100635 administered systemically (0.3 mg kg⁻¹, s.c.) or into the dorsal raphe (0.1 μg 0.5 μl⁻¹) in combination with 10 mg kg⁻¹ i.p. citalopram. The hypophagic effect of 10 mg kg⁻¹ i.p. citalopram alone was not significantly modified by SB206553.
6. Brain concentrations of citalopram and its metabolite desmethylcitalopram in rats pretreated with SB206553, WAY100635 and their combination were comparable to those of vehicle-pretreated rats, 90 min after citalopram injection.
7. The hypophagic effect of citalopram was potentiated by blocking 5-HT_1A receptors. Only the effect of the WAY100635/citalopram combination seemed to be partially mediated by central 5-HT_2C receptors.

     

The influence of antibodies to TNF-α and IL-1β on haemodynamic responses to the cytokines,and to lipopolysaccharide,in conscious rats

1. Male, Long Evans rats (350–450 g) were anaesthetized and had pulsed Doppler probes and intravascular catheters implanted to allow monitoring of regional (renal, mesenteric and hindquarters) haemodynamics in the conscious state. Our main objectives were to:- assess the effects of administering human recombinant tumour necrosis factor (TNF)-α and human recombinant interleukin-1 (IL-1)β, alone and together; determine the influence of pretreatment with a mixture of antibodies to TNF-α and IL-1β on responses to co-administration of the cytokines; ascertain if pretreatment with a mixture of the antibodies to TNF-α and IL-1β had any influence on the responses to lipopolysaccharide (LPS).
2. TNF-α (10, 100 and 250 μg kg⁻¹, in separate groups, n=3, 9 and 8, respectively) caused tachycardia (maximum Δ, +101±9 beats min⁻¹) and modest hypotension (maximum Δ, −10±2 mmHg), accompanied by variable changes in renal and mesenteric vascular conductance, but clear increases in hindquarters vascular conductance; only the latter were dose-related (maximum Δ, +6±6, +27±9, and +61±12% at 10, 100 and 250 μg kg⁻¹, respectively).
3. IL-1β (1, 10, and 100 μg kg⁻¹ in separate groups, n=8, 8 and 9, respectively) evoked changes similar to those of TNF-α (maximum Δ heart rate, +69±15 beats min⁻¹; maximum Δ mean blood pressure, −14±2 mmHg; maximum Δ hindquarters vascular conductance, +49±17%), but with no clear dose-dependency.
4. TNF-α (250 μg kg⁻¹) and IL-1β (10 μg kg⁻¹) together caused tachycardia (maximum Δ, +76±15 beats min⁻¹) and hypotension (maximum Δ, −24±2 mmHg) accompanied by increases in renal, mesenteric and hindquarters vascular conductances (+52±6%, +23±8%, and +52±11%, respectively). Thereafter, blood pressure recovered, in association with marked reductions in mesenteric and hindquarters vascular conductances (maximum Δ, −50±3% and −58±3%, respectively). Although bolus injection of LPS (3.5 mg kg⁻¹) caused an initial hypotension (maximum Δ, −27±11 mmHg) similar to that seen with co-administration of the cytokines, it did not cause mesenteric or hindquarters vasodilatation, and there was only a slow onset renal vasodilatation. The recovery in blood pressure following LPS was less than after the cytokines, and in the former condition there was no mesenteric vasoconstriction. By 24 h after co-administration of TNF-α and IL-1β or after bolus injection of LPS, the secondary reduction in blood pressure was similar (−16±2 and −13±3 mmHg, respectively), but in the former group the tachycardia (+117±14 beats min⁻¹) and increase in hindquarters vascular conductance (+99±21%) were greater than after bolus injection of LPS (+54±16 beats min⁻¹ and +43±9%, respectively).
5. Pretreatment with antibodies to TNF-α and IL-1β (300 mg kg⁻¹) blocked the initial hypotensive and mesenteric and hindquarters vasodilator responses to co-administration of the cytokines subsequently. However, tachycardia and renal vasodilatation were still apparent. Premixing antibodies and cytokines before administration prevented most of the effects of the latter, but tachycardia was still present at 24 h.
6. Pretreatment with antibodies to TNF-α and IL-1β before infusion of LPS (150 μg kg⁻¹ h⁻¹ for 24 h) did not affect the initial fall in blood pressure, but suppressed the hindquarters vasodilatation and caused a slight improvement in the recovery of blood pressure. However, pretreatment with the antibodies had no effect on the subsequent cardiovascular sequelae of LPS infusion.
7. The results indicate that although co-administration of TNF-α and IL-1β can evoke cardiovascular responses which, in some respects, mimic those of LPS, and although antibodies to the cytokines can suppress most of the cardiovascular effects of the cytokines, the antibodies have little influence on the haemodynamic responses to LPS, possibly because, during infusion of LPS, the sites of production and local action of endogenous cytokines, are not accessible to exogenous antibodies.

     

Role of Ca2+-activated K+ channels in acetylcholine-induced dilatation of the basilar artery in vivo

1. We tested the hypothesis that activation of large conductance calcium-activated potassium channels is involved in dilator responses of the basilar artery to acetylcholine in vivo. Using a cranial window in anaesthetized rats, we examined responses of the basilar artery to acetylcholine.
2. Topical application of acetylcholine (10⁻⁶ and 10⁻⁵ M) increased diameter of the basilar artery from 238±7 μm to 268±7 and 288±7 μm, respectively (P<0.05 vs. baseline diameter). Iberiotoxin (10⁻⁸ M), an inhibitor of large conductance calcium-activated potassium channels, did not affect baseline diameter of the basilar artery. In the presence of 10⁻⁸ M iberiotoxin, 10⁻⁶ and 10⁻⁵ M acetylcholine increased diameter of the basilar artery from 239±7 μm to 246±7 and 261±7 μm, respectively. Thus, iberiotoxin attenuated acetylcholine-induced dilatation of the basilar artery (P<0.05).
3. Sodium nitroprusside (10⁻⁷ and 10⁻⁶ M) increased diameter of the basilar artery from 242±9 μm to 310±12 and 374±13 μm, respectively (P<0.05 vs. baseline diameter). In the presence of iberiotoxin (10⁻⁸ M), sodium nitroprusside (10⁻⁷ and 10⁻⁶ M) increased diameter of the basilar artery from 243±6 μm to 259±9 and 311±12 μm, respectively. Thus, iberiotoxin attenuated dilator responses of the basilar artery to sodium nitroprusside (P<0.05).
4. Iberiotoxin partly inhibited dilator responses of the basilar artery to forskolin, a direct activator of adenylate cyclase, but did not affect vasodilatation produced by levcromakalim, a potassium channel opener.
5. These results suggest that dilator responses of the basilar artery to acetylcholine and sodium nitroprusside are mediated, in part, by activation of large conductance calcium-activated potassium channels. Because both acetylcholine and sodium nitroprusside have been shown to activate guanylate cyclase via nitric oxide, activation of large conductance calcium-activated potassium channels may be one of the major mechanisms by which cyclic GMP causes dilatation of the basilar artery in vivo.

     

Effect of nitrovasodilators and inhibitors of nitric oxide synthase on ischaemic and reperfusion function of rat isolated hearts

1. The functional role of the nitric oxide (NO)/guanosine 3′:5′-cyclic monophosphate (cyclic GMP) pathway in experimental myocardial ischaemia and reperfusion was studied in rat isolated hearts.
2. Rat isolated hearts were perfused at constant pressure with Krebs-Henseleit buffer for 25 min (baseline), then made ischaemic by reducing coronary flow to 0.2 ml min⁻¹ for 25 or 40 min, and reperfused at constant pressure for 25 min. Drugs inhibiting or stimulating the NO/cyclic GMP pathway were infused during the ischaemic phase only. Ischaemic contracture, myocardial cyclic GMP and cyclic AMP levels during ischaemia, and recovery of reperfusion mechanical function were monitored.
3. At baseline, heart rate was 287±12 beats min⁻¹, coronary flow was 12.8±0.6 ml min⁻¹, left ventricular developed pressure (LVDevP) was 105±4 mmHg and left ventricular end-diastolic pressure 4.6±0.2 mmHg in vehicle-treated hearts (control; n=12). Baseline values were similar in all treatment groups (P>0.05).
4. In normoxic perfused hearts, 1 μM N^G-nitro-L-arginine (L-NOARG) significantly reduced coronary flow from 13.5±0.2 to 12.1±0.1 ml min⁻¹ (10%) and LVDevP from 97±1 to 92±1 mmHg (5%; P<0.05, n=5).
5. Ischaemic contracture was 46±2 mmHg, i.e. 44% of LVDevP in control hearts (n=12), unaffected by low concentrations of nitroprusside (1 and 10 μM) but reduced to ∼30 mmHg (∼25%) at higher concentrations (100 or 1000 μM; P<0.05 vs control, n=6). Conversely, the NO synthase inhibitor L-NOARG reduced contracture at 1 μM to 26±3 mmHg (23%), but increased it to 63±4 mmHg (59%) at 1000 μM (n=6). Dobutamine (10 μM) exacerbated ischaemic contracture (81±3 mmHg; n=7) and the cyclic GMP analogue Sp-8-(4-p-chlorophenylthio)-3′,5′-monophosphorothioate (Sp-8-pCPT-cGMPS; 10 μM) blocked this effect (63±1 mmHg; P<0.05 vs dobutamine alone, n=5).
6. At the end of reperfusion, LVDevP was 58±5 mmHg, i.e. 55% of pre-ischaemic value in control hearts, significantly increased to ∼80% by high concentrations of nitroprusside (100 or 1000 μM) or L-NOARG at 1 μM, while a high concentration of L-NOARG (1000 μM) reduced LVDevP to ∼35% (P<0.05 vs control; n=6).
7. Ischaemia increased tissue cyclic GMP levels 1.8 fold in control hearts (P<0.05; n=12); nitroprusside at 1 μM had no sustained effect, but increased cyclic GMP ∼6 fold at 1000 μM; L-NOARG (1 or 1000 μM) was without effect (n=6). Nitroprusside (1 or 1000 μM) marginally increased cyclic AMP levels whereas NO synthase inhibitors had no effect (n=6).
8. In conclusion, the cardioprotective effect of NO donors, but not of low concentrations of NO synthase inhibitors may be due to their ability to elevate cyclic GMP levels. Because myocardial cyclic GMP levels were not affected by low concentrations of NO synthase inhibitors, their beneficial effect on ischaemic and reperfusion function is probably not accompanied by reduced formation of NO and peroxynitrite in this model.