Characterization of the peripheral action of neurokinins and neurokinin receptor selective agonists on the rat cardiovascular system

Summary The effects on mean arterial pressure (MAP) and heart rate (HR) of increasing doses (0.65–65 nmol/kg) of substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and selective agonists for neurokinin receptors were measured after intravenous (i. v.) injection in urethane anaesthetized rats. Neurokinins (NKs) elicited a vasodepressor effect with the following rank order of potency: SP (100%) > NKB (17.5%) > NKA (10%). The two undecapeptide NK-1 selective agonists, [Pro⁹, Met(O₂)¹¹]SP (787%) and [Sar⁹, Met(O₂)¹¹]SP (697%), evoked a significantly (P < 0.05) greater vasodepressor response than SP, while the potency of the octapeptide NK-1 selective agonist [-Ala⁴, Sar⁹, Met(O₂)¹¹]SP (4–11) (316%) was not significantly different from SP. Conversely, the NK-2 selective agonist NKA (4–10) (<2%) caused only a small effect. The vasodepressor effect elicited by [MePhe⁷]NKB (112%) and [-Asp⁴, MePhe⁷]NKB (4–10) (92%), two NK-3 selective agonists, were not significantly different from that of SP. Senktide (1,095%) is the most potent NK-3 agonist, and is significantly (P < 0.01) more potent than SP. No cross-desensitization, of the vasodepressor response, was observed between NK-1 and NK-3 selective agonists. I. V. injection of 32.5 nmol/kg of NKA, NKA (4–10) and [-Ala⁴, Sar⁹, Met(O₂)¹¹]SP (4–11) raised HR, while NKB and the NK-3 selective agonists produced a rapid and marked bradycardia. SP and the two undecapeptide, NK-1 selective agonists, produced an initial increase in HR and a latent long-lasting bradycardia. The bradycardia elicited by [Sar⁹, Met(O₂)¹¹]SP (32.5 nmol/kg) was blocked by methylatropine, hexamethonium, indomethacin and by treatment with capsaicin or compound 48/80. Although the bradycardia elicited by [-Asp⁴, MePhe⁷]NKB (4–10) (32.5 nmol/kg) was also blocked by hexamethonium, methylatropine, and by bilateral vagotomy, it remained unaffected after indomethacin, or in rats pretreated with either capsaicin or compound 48/80. The drop in MAP produced by the NK-1 and NK-3 agonists were reduced by hexamethonium, methylatropine and bilateral vagotomy (NK-3 agonist), but remained unaffected by indomethacin, capsaicin, and compound 48/80. The tachycardia to NKA (4–10) (65 nmol/kg) was blocked entirely by sotalol or metoprolol and potentiated by hexamethonium. Guanethidine and bilateral adrenalectomy (48 h) failed to affect the tachycardia induced by the agonist, whereas the combination of both treatments abolished the response. Rats sympathectomized with 6-hydroxydopamine (48 h) reduced the increase in HR to NKA (4–10) only at 1 min post-administration. These results support the hypothesis that the vasodepressor action of SP is mediated by the direct activation of NK-1 receptors on vascular endothelium. NKs can accelerate HR by releasing catecholamines through activation of NK-1 and/or NK-2 receptors, on noradrenergic fibers and adrenal chromaffn cells. Furthermore NKs can exert a depressor effect on the cardiovascular system by producing a vagal reflex (Bezold-Jarisch reflex), following either the activation of NK-3 receptors located on capsaicin-insensitive sensory fibers, or by promoting the release of prostaglandins from mast cells (via a non-receptor mechanism) which in turn will stimulate capsaicin-sensitive sensory fibers.     

Effects of neuropeptide Y on the cardiovascular system

The actions of neuropeptide Y (NPY) on the circulation is currently the subject of much research that has been stimulated by findings of its co-storage and co-operation with noradrenaline in sympathetic nerve fibres. Lars Edvinsson and colleagues describe the evidence that NPY is involved in the regulation of the cardiovascular system as a neuromodulator/neurotransmitter. This may open novel avenues for the treatment of, for example, hypertension and vasospastic disorders.     

Inhibitory action of (±)CP-96,345 on the cardiovascular responses to intrathecal substance P and neuropeptide K in the conscious freely moving rat

Summary (±)CP-96,345, a nonpeptide and highly selective NK-1 receptor antagonist, was tested acutely and chronically as an inhibitor of the cardiovascular responses induced by the intrathecal (i.t.) injection of substance P (SP) and neuropeptide K (NPK) in the conscious rat. When given at T-9 spinal cord level, NPK (0.65, 3.25 and 6.5 nmol) and SP (6.5, 16.25 and 32.5 nmol) produced increases in mean arterial pressure and heart rate. The cardiovascular responses to NPK were greater in intensity and duration than those produced by SP. The prior i.t. injection of (±)CP-96,345 (0.65 and 6.5 nmol, 15 min earlier) inhibited in a dose-dependent manner the pressor response and the tachycardia induced by 6.5 nmol SP while 65 nmol of the antagonist was required to reduce the effects of 3.25 nmol NPK. However, both the SP and NPK-induced cardiovascular changes were blocked 2 days after the Lt. injection of 6.5 nmol (±)CP-96,345. Five days after a single i.t. injection of 6.5 nmol (±)CP-96,345, the cardiovascular response to SP remained unaffected while that of NPK was partially attenuated. Moreover, (±)CP-96,345 was active as an antagonist when given ix. at the dose of 0.13 mg/kg. Conversely, (±)CP-96,345 failed to block the cardiovascular effect caused by the i.t. injection of 81 pmol bradykinin and did not produce any changes on resting blood pressure and heart rate when given alone either i.t. or L v. The results indicate that (±)CP-96,345 is a specific and long-acting antagonist which crosses the blood-brain barrier to block the action of SP and NPK in the spinal cord. Furthermore, these findings are consistent with the hypothesis that receptors of the NK-1 subtype mediate the cardiovascular responses evoked by the spinal action of neurokinins.Correspondence to R. Couture at the above address     

Prejunctional modulatory action of neuropeptide Y on peripheral terminals of capsaicin-sensitive sensory nerves.

1. We have determined the effect of neuropeptide Y (NPY) on motor responses produced by activation of capsaicin-sensitive primary afferents in the guinea-pig isolated left atria (reserpine-pretreatment, atropine in the bath) and bronchi (atropine and indomethacin in the bath) using capsaicin itself and electrical field stimulation as stimuli. 2. In both preparations, NPY inhibited or suppressed the response produced by electrical field stimulation while leaving the response to a submaximal concentration of capsaicin unaffected. 3. NPY had no effect on motor responses produced by a submaximal concentration of calcitonin gene-related peptide (atria) or neurokinin A (bronchi), the putative endogenous mediators of the responses produced by activation of the 'efferent' function of sensory fibres in these preparations. 4. We conclude that NPY exerts a prejunctional inhibitory action on transmitter release from peripheral endings of capsaicin-sensitive nerves. Failure of NPY to modulate responses activated by capsaicin provides further evidence for the existence of two independent modes of activation of the 'efferent' function of capsaicin-sensitive sensory nerves.     

Effect of neuropeptide Y on alpha 2-adrenoceptor-mediated cardiovascular responses in the pithed rat.

1. The effects of neuropeptide Y (NPY) on the cardiovascular responses induced by stimulation of pre and postjunctional alpha 2-adrenoceptors were studied in the pithed normotensive rat. 2. The increase in diastolic blood pressure induced by cumulative injection of xylazine (1-1000 micrograms kg-1, i.v.) were potentiated by NPY (5 micrograms kg-1) but not affected by a lower dose (0.75 micrograms kg-1) of this peptide. 3. Xylazine (1-100 micrograms kg-1) inhibited in a dose-dependent manner the tachycardia induced by continuous electrical stimulation (0.2 Hz, 2 ms, 60 V) of the spinal cord (C7-Thl). 4. NPY (5 micrograms kg-1 but not 0.75 micrograms kg-1) enhanced the inhibitory effect of xylazine on the tachycardia induced by electrical stimulation without having any direct effect on heart rate. 5. These results suggest that there may be a positive interaction between NPY receptors, postjunctional alpha 2-adrenoceptors and between NPY receptors and postjunctional alpha 2-adrenoceptors in the cardiovascular system of the rat.     

The effect of baclofen on the cardiovascular system of the rat.

1 The cardiovascular responses to baclofen were investigated in anaesthetized rats. 2 Low doses of baclofen (less than 5 X 10(-8) mol), given intravenously, produced a transient fall in blood pressure and heart rate. Higher doses (greater than 5 X 10(-7) mol) produced a marked and prolonged increase in blood pressure accompanied by a rise in heart rate and cutaneous arterial dilatation. 3 The pressor and heart rate responses exhibited tachyphylaxis, and were abolished by hexamethonium, cervical cord section, reserpine-treatment and by a combination of alpha- and beta-adrenoceptor antagonists. 4 It is concluded that the increases in blood pressure and heart rate produced by high doses of baclofen are of central sympathetic origin.     

Age and castration modulate the inhibitory action of neuropeptide Y on neurotransmission in the rat vas deferens.

The potency of neuropeptide Y (NPY) to inhibit the electrically induced contractions of the epididymal half of the vas deferens diminishes markedly with age, being at least 20 times lower in the adult than in the 26-day-old rat. Castration sensitizes the epididymal segment to NPY in a testosterone-reversible manner. [Pro34]NPY was 3 times less potent than NPY in prepubertal rats and inactive in castrated adults, while NPY-(13-36) had no effect in either group. In the prostatic half, NPY and its analogs were active in rats from all ages studied; the order of potency being NPY greater than [Pro34]NPY greater than NPY-(13-36). The sensitivity of the prostatic segment from adult rats to NPY was unchanged by castration or testosterone replacement therapy. The NPY content of the ductus increases during development being higher in the prostatic than in the epididymal half at all ages studied. Castration decreases the peptide content in the two segments and the effect is prevented by testosterone administration. The present investigation demonstrated that the effect of NPY on vas deferens neurotransmission is subject to regulation by sex steroids, which affects differently the response of the two segments of the ductus.     

ATP-sensitive K+ channels of the cardiovascular system

ATP-sensitive K+ (KATP) channels are inhibited by intracellular ATP and activated by intracellular nucleoside diphosphates, and thus provide a link between cellular metabolism and excitability. They are widely distributed in various tissues including heart and vasculature, and thus may play essential regulatory roles in the cardiovascular system. Furthermore, KATP channels are the targets of two important classes of drugs, i.e., the antidiabetic sulfonylureas which block the channels, and a series of vasorelaxants called "K+ channel openers" which tend to maintain the channels in an open conformation. Recently, the molecular structure of KATP channels has been clarified in various tissues including cardiovascular system to be a complex of at least two subunits, i.e. SUR and Kir6.0. The KATP channels in heart and vascular smooth muscle now appear to be the complexes of SUR2A/Kir6.2 and SUR2B/Kir6.1, respectively. Further works are now in progress to understand the molecular mechanisms responsible for the control of KATP channel function by intracellular nucleotides and drugs.     

Role of the endothelial system in Bay K 8644 enantiomer and nifedipine vasomodulator action in rat aorta.

The potential importance of the endothelial system in regulating the effects of (-)-Bay K 8644 (0.1 microM), (+)-Bay K 8644 (0.1 microM) and nifedipine (10 nM) on resting tension, on contractile responses to noradrenaline (NA) and Ca2+ (in a Ca(2+)-free high-K+ solution), and on basal, NA-induced and K(+)-induced 45Ca2+ uptake, was investigated in rat aorta rings. Mechanical removal of endothelium considerably potentiated the contractile response induced by NA in standard medium and by Ca2+ in Ca(2+)-free high-K+ (15 mM) medium, but did not modify the response induced by Ca2+ in Ca(2+)-free high-K+ (55 mM) medium or by NA in Ca(2+)-free medium. Furthermore, the basal 45Ca2+ uptake and that induced by NA (10 microM) or KCl (15 and 55 mM) were similar in endothelium-rubbed and intact rings. (-)-Bay K 8644 (0.1 microM) shifted the NA and Ca2+ concentration-response curves to the left with potentiation of the maximal contraction. However, (+)-Bay K 8644 (0.1 microM) and nifedipine (10 nM) caused a shift to the right, with depression of the maximal contraction. The NA concentration-response curves, and those of Ca2+ in Ca(2+)-free high-K+ (55 mM) medium, were affected by the drugs to similar extents, and were not modified by the presence or absence of endothelial cells. The drugs tested did not affect resting tension. Basal 45Ca2+ uptake was not modified by either nifedipine or the Bay K 8644 enantiomers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiovascular and behavioural effects of centrally administered neuropeptide K in the rat: receptor characterization.

1. The cardiovascular and behavioural responses to intracerebroventricularly (i.c.v.) administered neuropeptide K (NPK) were studied in conscious rats. The central effects of NPK were characterized by pretreatment (i.c.v.) with selective antagonists for the NK1 ((+/-)-CP 96345 and RP 67580), NK2 (SR 48968) and NK3 (R 487) receptors. 2. NPK (10-65 pmol) induced tachycardia and dose-dependent increases of mean arterial blood pressure. The cardiovascular responses reached a maximum within 3 min post-injection and lasted for more than 1 h. Concurrently, NPK produced dose-dependent increases of face washing, head scratching, grooming, walking and wet dog shakes. 3. A desensitization of most of the behavioural responses (except head scratching) but not of the cardiovascular response was shown when two consecutive injections of 25 pmol NPK were given 24 h apart. 4. Both the cardiovascular and behavioural responses (except the head scratching) to 25 pmol NPK were blocked by pre-administration (i.c.v.) of 6.5 nmol (+/-)-CP 96345 or RP 67580 given 5 min earlier. No inhibition of NPK responses was observed when 6.5 nmol SR 48968 or R 487 were used in a similar study. Additionally, NPK effects were significantly reduced 24 h after the prior injection of (+/-)-CP 96345 but not of RP 67580. 5. These results support the involvement of NK1 receptors in the cardiovascular and behavioural effects of i.c.v. NPK. Thus, this peptide may play a putative role in central cardiovascular regulation as it is the most potent endogenous tachykinin described centrally, to date.     

Effects of ketamine on the peripheral autonomic nervous system of the rat.

The effects of ketamine (2-(o-chlorophenyl) 2-methylaminocyclohexanone) (2-50 mg/kg) on the responses of the pithed rat arterial pressure, anococcygeus muscle and colon to selective stimulation of the spinal autonomic outflows were examined. Ketamine depressed the vasopressor response produced by stimulation of the lumbar sympathetic outflow in a dose-dependent manner but did not significantly affect the pressor response to intravenous noradrenaline (NA) administration. Ketamine depressed the motor responses of the anococcygeus to stimulation of the pre-ganglionic lumbar sympathetic outflow or to stimulation of post-ganglionic fibres in the sacral region in a dose-dependent manner, the response to preganglionic stimulation being relatively more sensitive to such depression. The anococcygeus response to NA was significantly potentiated with doses of ketamine of 20 mg/kg and 50 mg/kg. Ketamine depressed the motor response of the smooth muscle of the colon to stimulation of the sacral parasympathetic outflow in a dose-dependent manner and at lower doses than were required to produce an equivalent depression of the sympathetic responses in the other tissues. A comparison was made of the effects of ketamine and cocaine on the motor responses of the anococcygeus muscle in vitro to NA, carbachol and field stimulation. Both ketamine and cocaine produced a non-specific depression of all responses at high doses whereas cocaine but not ketamine produced a large potentiation of NA and motor nerve responses at lower doses. The results are discussed in relation to the hypothesis that ketamine might elevate blood pressure in conscious animals and man by potentiating vascular adrenergic responses.     

Characterization of vascular neuropeptide Y receptors.

1. In the present study we compared neuropeptide Y (NPY) and NPY-related analogues for their ability to activate or bind to vascular NPY receptors in four experimental set-ups. Previous results have suggested the existence of different receptor subtypes, Y1 receptors requiring full-length NPY (1-36) or [Pro34]-NPY, and Y2 receptors recognizing also N-terminally truncated forms of NPY but not [Pro34]-NPY. 2. NPY 1-36 and [Pro34]-NPY dose-dependently increased arterial pressure in the anaesthetized rat with a similar magnitude and potency. NPY 2-36 was much less potent than NPY 1-36. NPY 4-36 and NPY 11-36 were inactive even at a dose as high as 10 nmol kg-1. 3. NPY 1-36, [Pro34]-NPY, NPY 2-36 and NPY 5-36 concentration-dependently increased the coronary resistance in the Langendorff preparation of the rat. NPY 1-36 and [Pro34]-NPY were equipotent, while NPY 2-36 and NPY 5-36 were about 7 and 20 times less potent. At 0.3 microM, NPY 11-36, NPY 20-36 and NPY 22-36 induced a slight contraction while NPY 23-36 was inactive. 4. NPY 1-36, [Pro34]-NPY, NPY 2-36, NPY 4-36, NPY 5-36 and NPY 11-36 evoked concentration-dependent contractions in the isolated inferior caval vein of the rat and guinea-pig. [Pro34]-NPY was more potent than NPY 1-36. NPY 2-36 was equipotent with NPY 1-36, while NPY 4-36, NPY 5-36 and NPY 11-36 were approximately 30 times less potent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacology of neuropeptide Y receptor antagonists. Focus on cardiovascular functions

Neuropeptide Y is one of the most abundant mammalian neuropeptides identified to date. The possible actions of neuropeptide Y, that is co-localized and released with noradrenaline, as a sympathetic co-transmitter has attracted much attention during the last decade. In recent years, several non-peptide antagonists with high subtype selectivity for neuropeptide Y receptors have been introduced. With them, the status of neuropeptide Y as a sympathetic transmitter has been established, and so have profound cardiovascular effects mediated by neuropeptide Y Y(1) and Y(2) receptors. Significant release of neuropeptide Y occurs especially upon stronger sympathetic activation, and recent data suggest that the importance of neuropeptide Y seems enhanced in stress-related cardiovascular disorders. The true significance of neuropeptide Y has thus started to unfold, owing to the presence of the first generation of selective neuropeptide Y receptor antagonists. This review concerns the pharmacology of these agents, what we have learnt from them, and might find out in the future.     

The subdivision of beta-adrenoceptors in the cardiovascular system of the rat.

1 The antagonism by the beta-adrenoceptor blocking drugs, propranolol (non-selective) and practolol (beta-selective), of the cardiovascular actions of isoprenaline has been investigated in the rat. 2 All doses of practolol (0.1, 1 and 3 mg/kg) blocked the cardio-accelerator action of isoprenaline but only the largest dose blocked the vasodilator effect. 3 All doses of propranolol (0.01, 0.03 and 0.1 mg/kg) blocked the vasodilator effect of isoprenaline but only the largest dose diminished the tachycardia. 4 It is concluded that in the rat, as in other species, beta-adrenoceptors may be subdivided into beta 1 (cardiac) and beta 2 (peripheral vascular) types.     

Characterization and purification of the vitamin K1 2,3 epoxide reductases system from rat liver

The enzyme vitamin K1 2,3 epoxide reductase is responsible for converting vitamin K1 2,3 epoxide to vitamin K1 quinone thus completing the vitamin K cycle. The enzyme is also the target of inhibition by the oral anticoagulant, R,S-warfarin. Purification of this protein would enable the interaction of the inhibitor with its target to be elucidated. To date a single protein possessing vitamin K1 2,3 epoxide reductase activity and binding R,S-warfarin has yet to be purified to homogeneity, but recent studies have indicated that the enzyme is in fact at least two interacting proteins. We report on the attempted purification of the vitamin K1 2,3 epoxide reductase complex from rat liver microsomes by ion exchange and size exclusion chromatography techniques. The intact system consisted of a warfarin-binding factor, which possessed no vitamin K1 2,3 epoxide reductase activity and a catalytic protein. This catalytic protein was purified 327-fold and was insensitive to R,S-warfarin inhibition at concentrations up to 5 mM. The addition of the S-200 size exclusion chromatography fraction containing the inhibitor-binding factor resulted in the return of R,S-warfarin inhibition. Thus, to function normally, the rat liver endoplasmic reticulum vitamin K1 2,3 epoxide reductase system requires the association of two components, one with catalytic activity for the conversion of the epoxide to the quinone and the second, the inhibitor binding factor. This latter enzyme forms the thiol-disulphide redox centre that in the oxidized form binds R,S-warfarin.     

Characterization of the renal action of pranidipine in the rat

Although calcium antagonists elicit predominant dilation of afferent arterioles that might be associated with glomerular hypertension, there have been reported diverse observations demonstrating the effect of calcium antagonists on the progression of renal injury. The present study examined the effect of pranidipine (CAS 99522-79-9) on the renal microvascular tone in the isolated perfused hydronephrotic rat kidney, and the progression of renal insufficiency in subtotally nephrectomized spontaneously hypertensive rats. In the hydronephrotic kidney, angiotensin II caused marked constriction of both afferent and efferent arterioles. The subsequent addition of pranidipine (10 nmol/l, 100 nmol/l, 1 mumol/l) elicited dose-dependent afferent arteriolar dilation, with 97 +/- 3% reversal at 1 mumol/l. In contrast, efferent arterioles were resistant to pranidipine, with only 20 +/- 4% reversal at 1 mumol/l. In subtotally nephrectomized rats, 10-week treatment with pranidipine (3.0 mg/kg/day) markedly decreased blood pressure (from 270 +/- 6 to 158 +/- 8 mmHg) and improved renal histopathological changes, including glomerular and arteriolar sclerosis. Proteinuria was also less than than in the control rats (233 +/- 5 vs. 305 +/- 26 mg/day). Thus, although glomerular hypertension might develop as a consequence of preferential afferent arteriolar dilation, pranidipine actually improved the renal injury in subtotally nephrectomized SHR. These ostensibly discrepant observations could be attributed to the simultaneous reduction in blood pressure and the salutary actions of this agent mediated by non-hemodynamic mechanisms.