Short-term regulation of the Cl-/HCO3(-) exchanger in immortalized SHR proximal tubular epithelial cells

The present study evaluated the activity of Cl⁻/HCO₃⁻ exchanger and the abundance of Slc26a6 in immortalized renal proximal tubular epithelial (PTE) cells from the Wistar–Kyoto rat (WKY) and spontaneously hypertensive rat (SHR) and identified the signaling pathways that regulate the activity of the transporter. The affinity for HCO₃⁻ was identical in WKY and SHR PTE cells, but V_max values (in pH units/min) in SHR PTE cells (0.4016) were significantly higher than in WKY PTE cells (0.2304). The expression of Slc26a6 in SHR PTE cells was sevenfold that in WKY PTE cells. Dibutyryl-cAMP (db-cAMP) or forskolin, which increased endogenous cAMP, phorbol-12,13-dibutyrate (PDBu) and anisomycin, significantly (P < 0.05) increased the Cl⁻/HCO₃⁻ exchanger activity in WKY and SHR PTE cells to a similar extent. The stimulatory effects of db-cAMP and forskolin were prevented by the PKA inhibitor H89, but not by chelerythrine. The stimulatory effects of PDBu were prevented by both chelerythrine and SB 203580, but not by H89 or the MEK inhibitor PD 98059. The stimulatory effect of anisomycin was prevented by SB 203580, but not by chelerythrine. Increases in phospho-p38 MAPK by anisomycin were identical in WKY and SHR PTE cells, this being sensitive to SB 203580 but not to chelerythrine. It is concluded that SHR PTE cells, which overexpress the Slc26a6 protein, are endowed with an enhanced activity of the Cl⁻/HCO₃⁻ exchanger. The Cl⁻/HCO₃⁻ exchanger is an effector protein for PKA, PKC and p38 MAPK in both WKY and SHR PTE cells.     

Blowing off acid: a new tool to study Na+/HCO3- co-transport

Investigation of the physiological functions and possible pathological roles of Na(+)/HCO(3)(-) co-transport in the heart has been hampered by uncertainty over the molecular identity of cardiac Na(+)/HCO(3)(-) co-transporter(s) and the absence of selective pharmacological inhibitors. In their paper published in this issue, Ch'en and colleagues describe the extensive characterization of S0859 as a high-affinity inhibitor of Na(+)/HCO(3)(-) co-transport in cardiac myocytes (Ch'en et al., 2008). The availability of S0859 provides a powerful new tool to investigate the (patho)physiological significance of Na(+)/HCO(3)(-) co-transport in the heart and other tissues.     

Increased responsiveness to JNK1/2 mediates the enhanced H2O2-induced stimulation of Cl/HCO3 exchanger activity in immortalized renal proximal tubular epithelial cells from the SHR

We have previously demonstrated that exogenous H₂O₂ stimulates Cl⁻/HCO₃⁻ exchanger activity in immortalized renal proximal tubular epithelial (PTE) cells from both the Wistar-Kyoto (WKY) rat and the spontaneously hypertensive rat (SHR), this effect being more pronounced in SHR cells. The aim of the present study was to examine the mechanism of H₂O₂-induced stimulation of Cl⁻/HCO₃⁻ exchanger activity in WKY and SHR cells. It is now reported that the SHR PTE cells were endowed with an enhanced capacity to produce H₂O₂, comparatively with WKY cells and this was accompanied by a decreased expression of SOD2, SOD3, and catalase in SHR PTE cells. The stimulatory effect of H₂O₂ on the exchanger activity was blocked by SP600125 (JNK inhibitor), but not by U0126 (MEK1/2 inhibitor) or SB203580 (p38 inhibitor) in both cell lines. Basal JNK1 and JNK2 protein expression was higher in SHR PTE cells than in WKY PTE cells. H₂O₂ had no effect on p-JNK1/2 in WKY PTE cells over time. By contrast, H₂O₂ treatment resulted in a rapid and sustained increase in JNK1/2 phosphorylation in SHR PTE cells, which was completely abolished by apocynin. Treatment of SHR PTE cells with apocynin significantly decreased the H₂O₂-induced stimulation of Cl⁻/HCO₃⁻ exchanger activity. It is concluded that H₂O₂-induced stimulation of Cl⁻/HCO₃⁻ exchanger activity is regulated by JNK1/2, particularly by JNK2, in SHR PTE cells. The imbalance between oxidant and antioxidant mechanisms in SHR PTE cells enhances the response of JNK1/2 to H₂O₂, which contributes to their increased sensitivity to H₂O₂.     

Role of alpha1-adrenoceptor subtypes in the effects of methylenedioxy methamphetamine (MDMA) on body temperature in the mouse

BACKGROUND AND PURPOSE: We have investigated the ability of alpha(1)-adrenoceptor antagonists to affect the hyperthermia produced by methylenedioxy methamphetamine (MDMA) in conscious mice. EXPERIMENTAL APPROACH: Mice were implanted with temperature probes under ether anaesthesia and allowed 2 weeks recovery. MDMA (20 mg kg(-1)) was administered subcutaneously 30 min after vehicle or test antagonist or combination of antagonists and effects on body temperature monitored. KEY RESULTS: Following vehicle, MDMA produced a hyperthermia, reaching a maximum increase of 1.8 degrees C at 140 min. Prazosin (0.1 mg kg(-1)) revealed an early significant hypothermia to MDMA of -1.94 degrees C. The alpha(1A)-adrenoceptor antagonist RS 100329 (0.1 mg kg(-1)), or the alpha(1D)-adrenoceptor antagonist BMY 7378 (0.5 mg kg(-1)) given alone, did not reveal a hypothermia to MDMA, but the combination of the two antagonists revealed a significant hypothermia to MDMA. The putative alpha(1B)-adrenoceptor antagonist cyclazosin (1 mg kg(-1)) also revealed a significant hypothermia to MDMA, but actions of cyclazosin at the other alpha(1)-adrenoceptor subtypes cannot be excluded. CONCLUSIONS AND IMPLICATIONS: More than one subtype of alpha(1)-adrenoceptor is involved in a component of the hyperthermic response to MDMA in mouse, probably both alpha(1A)- and alpha(1D)-adrenoceptors, and removal of this alpha(1)-adrenoceptor-mediated component reveals an initial hypothermia.     

Identification of the alpha1L-adrenoceptor in rat cerebral cortex and possible relationship between alpha1L- and alpha1A-adrenoceptors

BACKGROUND AND PURPOSE: In addition to alpha1A, alpha1B and alpha1D-adrenoceptors (ARs), putative alpha1L-ARs with a low affinity for prazosin have been proposed. The purpose of the present study was to identify the alpha1A-AR and clarify its pharmacological profile using a radioligand binding assay. EXPERIMENTAL APPROACH: Binding experiments with [3H]-silodosin and [3H]-prazosin were performed in intact tissue segments and crude membrane preparations of rat cerebral cortex. Intact tissue binding assays were also conducted in rat tail artery. KEY RESULTS: [3H]-silodosin at subnanomolar concentrations specifically bound to intact tissue segments and membrane preparations of rat cerebral cortex at the same density (approximately 150 fmol mg(-1) total tissue protein). The binding sites in intact segments consisted of alpha1A and alpha1L-ARs that had different affinities for prazosin, while the binding sites in membranes showed an alpha1A-AR-like profile having single high affinity for prazosin. [3H]-prazosin also bound at subnanomolar concentrations to alpha1A and alpha1B-ARs but not alpha1L-ARs in cerebral cortex; the binding densities being approximately 200 and 290 fmol mg(-1) protein in the segments and the membranes, respectively. In the segments of tail artery, [3H]-silodosin only recognized alpha1A-ARs, whereas [3H]-prazosin bound to alpha1A and alpha1B-ARs. CONCLUSIONS AND IMPLICATIONS: The present study clearly reveals the presence of alpha1L-ARs as a pharmacologically distinct entity from alpha1A and alpha1B-ARs in intact tissue segments of rat cerebral cortex but not tail artery. However, the alpha1L-ARs disappeared after tissue homogenization, suggesting their decomposition and/or their pharmacological profile changes to that of alpha1A-ARs.     

Identification of alpha 1L-adrenoceptor in mice and its abolition by alpha 1A-adrenoceptor gene knockout

Background and purpose:

The α_1L-adrenoceptor has pharmacological properties that distinguish it from three classical α₁-adrenoceptors (α_1A, α_1B and α_1D). The purpose of this was to identify α_1L-adrenoceptors in mice and to examine their relationship to classical α₁-adrenoceptors.

Experimental approach:

Radioligand binding and functional bioassay experiments were performed on the cerebral cortex, vas deferens and prostate of wild-type (WT) and α_1A-, α_1B- and α_1D-adrenoceptor gene knockout (AKO, BKO and DKO) mice.

Key results:

The radioligand [³H]-silodosin bound to intact segments of the cerebral cortex, vas deferens and prostate of WT, BKO and DKO but not of AKO mice. The binding sites were composed of two components with high and low affinities for prazosin or RS-17053, indicating the pharmacological profiles of α_1A-adrenoceptors and α_1L-adrenoceptors. In membrane preparations of WT mouse cortex, however, [³H]-silodosin bound to a single population of prazosin high-affinity sites, suggesting the presence of α_1A-adrenoceptors alone. In contrast, [³H]-prazosin bound to two components having α_1A-adrenoceptor and α_1B-adrenoceptor profiles in intact segments of WT and DKO mouse cortices, but AKO mice lacked α_1A-adrenoceptor profiles and BKO mice lacked α_1B-adrenoceptor profiles. Noradrenaline produced contractions through α_1L-adrenoceptors with low affinity for prazosin in the vas deferens and prostate of WT, BKO and DKO mice. However, the contractions were abolished or markedly attenuated in AKO mice.

Conclusions and implications:

α_1L-Adrenoceptors were identified as binding and functional entities in WT, BKO and DKO mice but not in AKO mice, suggesting that the α_1L-adrenoceptor is one phenotype derived from the α_1A-adrenoceptor gene.     

Beta3-adrenoceptor agonist stimulation of the Na+, K+ -pump in rat skeletal muscle is mediated by beta2- rather than beta3-adrenoceptors

BACKGROUND AND PURPOSE: In cardiac muscle, BRL 37344, a selective beta3-adrenoceptor agonist, activates the Na+, K+ -pump via NO signalling. This study investigated whether BRL 37344 also activates the Na+, K+ -pump via beta3-adrenoceptors in skeletal muscle. EXPERIMENTAL APPROACH: Isolated rat soleus muscles were incubated between 1 and 60 min in buffer. Intracellular Na+, K+ content and Na+, K+ -pump activity were measured using flame photometry and ouabain-suppressible 86Rb+ uptake, respectively. Additional muscles were mounted on force transducers and stimulated (60 Hz for 2 s) every 10 min. KEY RESULTS: BRL 37344 (10(-8) -10(-5) M) induced a concentration- and time-dependent reduction in intracellular Na+, and increased ouabain-suppressible 86Rb+ uptake by up to 112%. BRL 37344-induced reductions in intracellular Na+ were blocked by the beta1/beta2-adrenoceptor antagonist, nadolol (10(-7) M), and the beta2-adrenoceptor antagonist, ICI 118,551 (10(-7) -10(-5) M), but not by beta3- or beta1-adrenoceptor antagonists, SR 59230A (10(-7) M) and CGP 20712A (10(-7) -10(-5) M), respectively. Another beta3-adrenoceptor agonist, CL 316,243, did not alter intracellular Na+. BRL 37344-induced reductions in intracellular Na+ were not blocked by L-NAME, an NOS inhibitor, or ODQ, a guanylyl cyclase inhibitor. The NO donors, SNP and SNAP, did not alter intracellular Na+. BRL 37344 rapidly recovered force in muscles depressed by high [K+]o, an effect that was blocked by nadolol, but not L-NAME. CONCLUSIONS AND IMPLICATIONS: In rat soleus muscle, the beta3-adrenoceptor agonist BRL 37344 stimulated the Na+, K+ -pump via beta2-adrenoceptors. A more selective beta3-adrenoceptor agonist did not affect Na+, K+ homeostasis in skeletal muscle. NO did not seem to mediate Na+, K+ -pump stimulation in skeletal muscle.     

Action potential shortening through the putative β4-adrenoceptor in ferret ventricle: comparison with β1- and β2-adrenoceptor-mediated effects

The electrophysiological responses to (−)-CGP 12177 ((−)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one), an agonist for the putative β₄-adrenoceptor, were investigated on isolated perfused ferret hearts paced at 100 min⁻¹ and compared to those of (−)-noradrenaline and (−)-adrenaline, mediated through β₁- and β₂-adrenoceptors respectively. The three agonists decreased ventricular monophasic action potential duration but prolonged the action potential plateau; β₃-adrenoceptor-selective agonists had no effect. (−)-CGP 12177 was the most potent, but (−)-noradrenaline the most efficacious; both agonists caused ventricular extra-systoles. Because only (−)-noradrenaline but not (−)-CGP 12177 elicited shortening of the refractory period, the mechanism of arrhythmias mediated through β₁- and putative β₄-adrenoceptors may be different.     

alpha(1)-Adrenoceptors in the lateral septal area modulate food intake behaviour in rats

Background and purpose:

Control of food intake is a complex behaviour which involves many interconnected brain structures. The present work assessed if the noradrenergic system in the lateral septum (LS) was involved in the feeding behaviour of rats.

Experimental approach:

In the first protocol, the food intake of rats was measured. Then non-food-deprived animals received either 100 nL of 21 nmol of noradrenaline or vehicle unilaterally in the LS 10 min after local 10 nmol of WB4101, an α₁-adrenoceptor antagonist, or vehicle. In the second protocol, different doses of WB4101 (1, 10 or 20 nmol in 100 nL) were microinjected bilaterally into the LS of rats, deprived of food for 18 h and food intake was compared to that of satiated animals.

Key results:

One-sided microinjection of noradrenaline into the LS of normal-fed rats evoked food intake, compared with vehicle-injected control animals, which was significantly reduced by α₁-adrenoceptor antagonism. In a further investigation, food intake was significantly higher in food-deprived animals, compared to satiated controls. Pretreatment of the LS with WB4101 reduced food intake in only food-deprived animals in a dose-related manner, suggesting that the LS noradrenergic system was involved in the control of food intake.

Conclusion and implications:

Activation by local microinjection of noradrenaline of α₁-adrenoceptors in the LS evoked food intake behaviour in rats. In addition, blockade of the LS α₁-adrenoceptors inhibited food intake in food-deprived animals, suggesting that the LS noradrenergic system modulated food intake behaviour and satiation.     

alpha1beta2delta, a silent GABAA receptor: recruitment by tracazolate and neurosteroids

BACKGROUND AND PURPOSE: This study investigated the alpha(1)beta(2)delta isoform of the GABA(A) receptor that is presumably expressed in the forebrain. The functional and pharmacological properties of this receptor combination are largely unknown. EXPERIMENTAL APPROACH: We expressed alpha(1)beta(2)delta GABA(A) receptors in Xenopus laevis oocytes. GABA-activated currents, in the presence and absence of modulators, were recorded using the two-electrode voltage clamp technique. KEY RESULTS: The alpha(1)beta(2)delta isoform of the GABA(A) receptor exhibited an extremely small GABA-mediated current. Tracazolate increased the current amplitude evoked by a half-maximal concentration (EC(50)) of GABA by 59-fold. The maximum current was increased 23-fold in the presence of a saturating GABA concentration. Concomitant with the increase in the maximum, was a 4-fold decrease in the EC(50). Finally, a mutation in the second transmembrane domain of the delta subunit that increases receptor efficacy (L286S), eliminated the increase in the maximum GABA-activated current. The endogenous neurosteroid, tetrahydrodeoxycorticosterone (THDOC), also decreased the EC(50) and increased the maximum current amplitude, although to a lesser degree than that of tracazolate. CONCLUSIONS AND IMPLICATIONS: Taken all together, these findings indicate that the small GABA-mediated currents in the absence of the modulator are due to a low efficacy for activation. In the absence of modulators, alpha(1)beta(2)delta GABA receptors would be effectively silent and therefore contribute little to inhibition in the CNS. In the presence of tracazolate or endogenous neurosteroids however, this particular receptor isoform could exert a profound inhibitory influence on neuronal activity.     

Testosterone protects rat hearts against ischaemic insults by enhancing the effects of alpha(1)-adrenoceptor stimulation

BACKGROUND AND PURPOSE: Testosterone alleviates symptoms in patients with ischaemic heart disease. Androgen receptors are present in the heart, and testosterone upregulates gene expression of cardiac beta(1)-adrenoceptors. We hypothesize that testosterone may confer cardioprotection by interacting with adrenoceptors.EXPERIMENTAL APPROACH: In isolated perfused hearts and ventricular myocytes from orchidectomized rats without or with testosterone (200 microg/100 g) replacement, we first determined the effect of ischaemia/reperfusion in the presence of noradrenaline (10(-7) M). Then we determined the contribution of interactions between testosterone and alpha(1)- or beta(1)-adrenoceptors in cardiac injury/protection (infarct size, release of lactate dehydrogenase, viability of myocytes, recovery of contractile function and incidence of arrhythmias) upon ischaemia/reperfusion by pharmacological manipulation using selective adrenoceptor agonists (alpha(1)-adrenoceptor agonist: phenylephrine 10(-6) M; non-selective beta-adrenoceptor agonist: isoprenaline 10(-7) M) and antagonists (alpha(1): prazosin or benoxathian 10(-6) M; beta(1): CGP 20712A 5 x 10(-7) M). We also determined the expression of alpha(1) and beta(1)-adrenoceptor in the hearts from rats with and without testosterone.KEY RESULTS: Testosterone reduced injury induced by ischaemia/reperfusion and noradrenaline. This was achieved by enhancing the beneficial effect of alpha(1)-adrenoceptor stimulation, which was greater than the deleterious effect of beta(1)-adrenoceptor stimulation (also enhanced by testosterone). The effects of testosterone were abolished or attenuated by blockade of androgen receptors. Testosterone also enhanced the expression of alpha(1A) and beta(1)-adrenoceptor.CONCLUSIONS AND IMPLICATIONS: Testosterone conferred cardioprotection by upregulating the cardiac alpha(1)-adrenoceptor and enhancing the effects of stimulation of this adrenoceptor. The effect of testosterone was at least partly mediated by androgen receptors.     

Influence of combined hypertension and renal failure on functional alpha(1)-adrenoceptor subtypes in the rat kidney

BACKGROUND AND PURPOSE: This study investigated whether the alpha(1)-adrenoceptor responsiveness of the renal vasculature was altered in the state of hypertension combined with renal failure. EXPERIMENTAL APPROACH: Male spontaneously hypertensive rats (SHR) received cisplatin (5 mg kg(-1) i.p.) to induce renal failure. Seven days later, the rats were anaesthetized and the reductions in renal blood flow (RBF) to electrical renal nerve stimulation (RNS) and intrarenal administration of three adrenoceptor agonists (noradrenaline, phenylephrine and methoxamine) were determined before and after amlodipine, 5-methylurapidil, chloroethylclonidine or BMY 7378. KEY RESULTS: In renal failure SHR (RFSHR), RBF and creatinine clearance were significantly reduced (approximately 70%), while urine output and fractional sodium excretion were four and twenty-fold higher, respectively, compared to SHR. Vasoconstrictions induced by RNS or the adrenoceptor agonists were greater in RFSHR than SHR, and these responses were blunted by 5-methylurapidil, BMY 7378 and amlodipine in the SHR, while chloroethylclonidine had no effect. In the RFSHR, all renal vasoconstrictions were reduced by amlodipine and BMY 7378 but 5-methylurapidil attenuated those caused by RNS, noradrenaline and methoxamine while those to phenylephrine were enhanced. Chloroethylclonidine potentiated renal vasoconstrictor responses to methoxamine and phenylephrine but not RNS or noradrenaline in RFSHR. CONCLUSIONS AND IMPLICATIONS: These findings suggest alpha(1A)- and alpha(1D)-adrenoceptors mediated the renal vasoconstrictor responses in SHR and RFSHR. In the RFSHR, other alpha(1)-adrenoceptor subtypes, for example, alpha(1B)-adrenoceptors appeared to play a greater role.     

Relevance of anaesthesia for dofetilide-induced torsades de pointes in alpha1-adrenoceptor-stimulated rabbits

BACKGROUND AND PURPOSE: No information is available concerning the effects of anaesthetics in the most frequently used in vivo pro-arrhythmia model. Accordingly, in this study we examined the effect of pentobarbital, propofol or alpha-chloralose anaesthesia on the pro-arrhythmic activity of the class III anti-arrhythmic dofetilide in alpha(1)-adrenoceptor-stimulated rabbits. EXPERIMENTAL APPROACH: Rabbits anaesthetized intravenously with pentobarbital, propofol or alpha-chloralose were infused simultaneously with the alpha(1)-adrenoceptor agonist phenylephrine (15 microg kg(-1) min(-1), i.v.) and dofetilide (0.04 mg kg(-1) min(-1), i.v.). The electrocardiographic QT interval, the T (peak)-T (end) interval and certain QT variability parameters were measured. The heart rate variability and the baroreflex sensitivity were utilized to assess the vagal nerve activity. The spectral power of the systolic arterial pressure was calculated in the frequency range 0.15-0.5 Hz to assess the sympathetic activity. KEY RESULTS: Pentobarbital considerably reduced, whereas propofol did not significantly affect the incidence of dofetilide-induced torsades de pointes (TdP) as compared with the results with alpha-chloralose (40% (P=0.011) and 70% (P=0.211) vs 100%, respectively). In additional experiments, neither doubling of the rate of the dofetilide infusion nor tripling of the rate of phenylephrine infusion elevated the incidence of TdP to the level seen with alpha-chloralose. None of the repolarization-related parameters predicted TdP. The indices of the parasympathetic and sympathetic activity were significantly depressed in the alpha-chloralose and propofol anaesthesia groups. CONCLUSIONS AND IMPLICATIONS: In rabbits, anaesthetics may affect drug-induced TdP genesis differently, which must be considered when results of different studies are compared.     

Phosphodiesterase-4 blunts inotropism and arrhythmias but not sinoatrial tachycardia of (-)-adrenaline mediated through mouse cardiac beta(1)-adrenoceptors

BACKGROUND AND PURPOSE: beta(1) and beta(2)-adrenoceptors coexist in murine heart but beta(2)-adrenoceptor-mediated effects have not been detected in atrial and ventricular tissues, possibly due to marked phosphodiesterase (PDE) activity. We investigated the influence of the PDE3 inhibitor cilostamide and PDE4 inhibitor rolipram on the effects of (-)-adrenaline in three regions of murine heart. EXPERIMENTAL APPROACH:(-)-Adrenaline-evoked cardiostimulation was compared on sinoatrial beating rate, left atrial and right ventricular contractile force in isolated tissues from 129SvxC57B1/6 cross mice. Ventricular arrhythmic contractions were also assessed. KEY RESULTS: Both rolipram (1 microM) and cilostamide (300 nM) caused transient sinoatrial tachycardia but neither enhanced the chronotropic potency of (-)-adrenaline. Rolipram potentiated 19-fold (left atrium) and 7-fold (right ventricle) the inotropic effects of (-)-adrenaline. (-)-Adrenaline elicited concentration-dependent ventricular arrhythmias that were potentiated by rolipram. All effects of (-)-adrenaline were antagonized by the beta(1)-adrenoceptor-selective antagonist CGP20712A (300 nM). Cilostamide (300 nM) did not increase the chronotropic and inotropic potencies of (-)-adrenaline, but administered jointly with rolipram in the presence of CGP20712A, uncovered left atrial inotropic effects of (-)-adrenaline that were prevented by the beta(2)-adrenoceptor-selective antagonist ICI118551.CONCLUSIONS AND IMPLICATIONS: PDE4 blunts the beta(1)-adrenoceptor-mediated effects of (-)-adrenaline in left atrium and right ventricle but not in sinoatrial node. Both PDE3 and PDE4 reduce basal sinoatrial rate in a compartment distinct from the beta(1)-adrenoceptor compartment. PDE3 and PDE4, acting in concert, prevent left atrial beta(2)-adrenoceptor-mediated inotropy. PDE4 partially protects the right ventricle against (-)-adrenaline-evoked arrhythmias.     

Native profiles of alpha(1A)-adrenoceptor phenotypes in rabbit prostate

Background and purpose:

α₁-Adrenoceptors in the rabbit prostate have been studied because of their controversial pharmacological profiles in functional and radioligand binding studies. The purpose of the present study is to determine the native profiles of α₁-adrenoceptor phenotypes and to clarify their relationship.

Experimental approach:

Binding experiments with [³H]-silodosin and [³H]-prazosin were performed using intact tissue segments and crude membrane preparations of rabbit prostate and the results were compared with α₁-adrenoceptor-mediated prostate contraction.

Key results:

[³H]-Silodosin at subnanomolar concentrations bound specifically to intact tissue segments of rabbit prostate. However, [³H]-prazosin at the same range of concentrations failed to bind to α₁-adrenoceptors of intact segments. Binding sites of [³H]-silodosin in intact segments were composed of α_1L phenotype with low affinities for prazosin (pKi=7.1), 5-methyurapidil and N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-α,α-dimethyl-1H-indole-3-ethamine hydrochloride (RS-17053), and α_1A-like phenotype with moderate affinity for prazosin (pKi=8.8) but high affinity for 5-methyurapidil and RS-17053. In contrast, both radioligands bound to a single population of α₁-adrenoceptors in the membrane preparations at the same density with a subnanomolar affinity, showing a typical profile of ‘classical'' α_1A-adrenoceptors (pKi for prazosin=9.8). The pharmacological profile of α₁-adrenoceptor-mediated prostate contraction was in accord with the α_1L phenotype observed by intact segment binding approach.

Conclusions and implications:

Three distinct phenotypes (α_1L and α_1A-like phenotypes in the intact segments and a classical α_1A phenotype in the membranes) with different affinities for prazosin were detected in rabbit prostate. It appears that the three phenotypes are phenotypic subtypes of α_1A-adrenoceptors, but are not genetically different subtypes.     

The Cl-HCO3- exchanger slows the recovery of acute pHi changes in rat mast cells

The CO(2)/HCO(3)(-) buffering system is one of the main mechanisms implicated in cytosolic pH (pH(i)) regulation. We studied this pH(i)-regulatory system in rat mast cells using a fluorescent dye. Mast cells had a more alkaline pH(i) in the presence of HCO(3)(-) than in its absence. The recovery from an acid load was faster in HCO(3)(-)-free conditions than in HCO(3)(-)-containing media. In HCO(3)(-)-buffered conditions the increase of the recovery rate of an acidification in 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-incubated cells suggested the implication of a Na(+)-independent Cl(-)/HCO(3)(-) exchanger. This HCO(3)(-) transport acidified the cytosol and was also partially responsible for the recovery of intracellular alkalinizations. Moreover, regulation of the recovery rate of an acidification by protein kinase C and calcium signaling pathways depended on the presence or absence of HCO(3)(-). The presence of HCO(3)(-) limits the recovery of acute intracellular acidifications probably through the Na(+)-independent Cl(-)/HCO(3)(-) exchanger and modulates the regulation of pH(i) by protein kinase C and calcium.     

Role of the cation-chloride-cotransporters in the circadian system

The circadian system plays an immense role in controlling physiological processes in our body. The suprachiasmatic nucleus (SCN) supervises this system, regulating and harmonising the circadian rhythms in our body. Most neurons present in the SCN are GABAergic neurons. Although GABA is considered the main inhibitory neurotransmitter of the CNS, recent studies have shown that excitatory responses were recorded in this area. These responses are enabled by an increase in intracellular chloride ions [Cl⁻]_i levels. The chloride (Cl⁻) levels in GABAergic neurons are controlled by two solute carrier 12 (SLC12) cation-chloride-cotransporters (CCCs): Na⁺/K⁺/Cl⁻ co-transporter (NKCC1) and K⁺/Cl⁻ co-transporter (KCC2), that respectively cause an influx and efflux of Cl⁻. Recent works have found altered expression and/or activity of either of these co-transporters in SCN neurons and have been associated with circadian rhythms. In this review, we summarize and discuss the role of CCCs in circadian rhythms, and highlight these recent advances which attest to CCC's growing potential as strong research and therapeutic targets.     

Phosphodiesterase isozymes involved in regulation of HCO3- secretion in isolated mouse duodenum in vitro

We examined the effects of various isozyme-selective PDE inhibitors on HCO(3)(-) secretion in the mouse duodenum in vitro and investigated which type(s) of phosphodiesterase (PDE) isozymes are involved in the response to PGE(2) and NO. The duodenal mucosa of male DDY mice was stripped of the muscle layer and mounted on an Ussing chamber, and HCO(3)(-) secretion was measured at pH 7.0 by a pH-stat method using 2mM HCl. Both PGE(2) and NOR-3 (NO donor) increased HCO(3)(-) secretion in the mouse duodenum in vitro, and the response to PGE(2) was inhibited by both EP3 and EP4 antagonists but not EP1 antagonist, while that to NOR-3 was inhibited by methylene blue. IBMX, a nonselective PDE inhibitor, significantly increased basal HCO(3)(-) secretion and potentiated the responses to both PGE(2) and NOR-3. Likewise, vinpocetine (PDE1 inhibitor) and cilostamide (PDE3 inhibitor) also increased the basal secretion at high doses and potentiated the HCO(3)(-) response to PGE(2) at doses that had no effect by themselves on the basal secretion. By contrast, the HCO(3)(-) stimulatory action of NOR-3 was significantly potentiated by vinpocetine but not cilostamide. Inhibitors of other PDE subtypes had no effect on the HCO(3)(-) secretion under basal or stimulated conditions. Both PDE1 and PDE3 mRNAs were expressed in the duodenal mucosa. These results suggested that PDE1 and PDE3 are involved in the regulation of duodenal HCO(3)(-) secretion and that the response to PGE(2) is associated with both PDE1 and PDE3, while the response to NO is mainly modulated by PDE1.     

Cardiovascular effects of activation of central alpha7 and alpha4beta2 nAChRs: a role for vasopressin in anaesthetized rats

BACKGROUND AND PURPOSE: Central application of nicotine causes the release of vasopressin and affects blood pressure. Involvement of the 5 neuronal nicotinic receptor groups, alpha2(*)-alpha7(*) in these effects is unknown. The availability of selective agonists for alpha7 (PSAB-OFP) and alpha4beta2 (TC-2559) nACh receptors allowed their role to be investigated. EXPERIMENTAL APPROACH: Recordings were made of arterial blood pressure, heart rate and renal sympathetic nerve activity in anaesthetized male rats with neuromuscular blockade and artificial respiration. Effects of the agonists, PSAB-OFP (1-10 micromol kg(-1)) and TC-2559 (1-10 micromol kg(-1)) on these variables given intracerebroventricularly (i.c.v.) and intracisternally (i.c.) in the presence or absence of the antagonists, DhbetaE (10 micromol kg(-1)) and MLA (0.5 micromol kg(-1)), for the appropriate nicotinic receptor subtypes, respectively, and a V(1) receptor antagonist, given i.v. or centrally, were investigated. KEY RESULTS: Both agonists given i.c.v. caused a delayed rise in blood pressure and renal nerve activity which could be blocked only with the appropriate antagonist. The agonists had an earlier onset of action when given i.c., favouring the brainstem as the major site of action. The effects of these agonists were also attenuated by the V(1) receptor antagonist given i.v. and blocked when this antagonist was given centrally. Antagonists had no effect on baseline variables.CONCLUSIONS AND IMPLICATIONS: Activation of alpha4beta2 and alpha7 receptors in the brainstem is mainly responsible for the cardiovascular effects of activating these receptors, which have a similar profile of action. These actions, although independent, are mediated by the central release of vasopressin.     

Characterization of the non-adrenergic/non-cholinergic response to perivascular nerve stimulation in the double-perfused mesenteric bed of the mouse

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide (CGRP), a capsaicin-sensitive neuromodulator of splanchnic vascular tone in several animal species, remains poorly investigated in mouse models. We therefore assessed whether endogenous CGRP is a non-adrenergic/non-cholinergic (NANC) neuromodulator in the mesenteric vascular bed of the mouse. EXPERIMENTAL APPROACH: Arterial and venous changes in perfusion pressure in response to perivascular nerve stimulation (PNS) were monitored in the mouse mesenteric bed under basal conditions or precontracted with KCl (artery) or U46619 (vein) in circuits pretreated with guanethidine, atropine, indomethacin and prazosin. Arterial responses to NANC were also characterized with a CGRP1 antagonist, halphaCGRP8-37. Finally, the PNS-induced release of arterial CGRP was measured by enzyme immunoassay. KEY RESULTS: HalphaCGRP8-37 enhanced PNS-induced arterial increases in perfusion pressure under basal tone. PNS-induced stimulation of NANC triggered an halphaCGRP8-37 or capsaicin- sensitive reduction in perfusion pressure of the pre-contracted arterial bed only. Chemical removal of the endothelium inhibited PNS- and halphaCGRP- induced reduction in perfusion pressure in the arterial mesenteric bed. Responses to NANC nerves were reduced by guanylate and adenylate cyclase inhibitors (1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one (ODQ)) and [9-(tetrahydro-2-furanyl)-9H-purin-6-amine] (SQ 22,536), respectively. A neuronal NOS inhibitor (7-nitroindazole; 7-NI) also enhanced the response to NANC in vessels from wild-type, eNOS KO but not iNOS KO mice. Finally, PNS enhanced the release of immunoreactive CGRP from the perfused arterial mesenteric bed. CONCLUSIONS AND IMPLICATIONS: Our study demonstrates a role for CGRP in the NANC-dependent reduction in perfusion pressure of the arterial but not venous mesenteric bed of the mouse.