Effects of nimodipine, Bay K 8644 and pinacidil on alpha 1- and alpha 2-adrenoceptor-mediated vasoconstriction in human hand veins

The effects of nimodipine, Bay K 8644 and pinacidil, three drugs interfering with transmembrane Ca2+ fluxes in different ways, were investigated in isolated human hand veins. Their ability to influence the concentration-response relationship for noradrenaline (NA) was assessed in the absence and presence of prazosin or rauwolscine. The contractile response to NA was almost abolished in Ca2+ -free medium. Nimodipine and pinacidil depressed the NA concentration-response curve both in the absence and presence of alpha-adrenoceptor blockers. The NA response was only partially inhibited by nimodipine, indicating that NA may activate nimodipine-insensitive influx pathways, presumably receptor-operated calcium channels. Pinacidil inhibited the contractile response to 124 mM K+ and reduced the NA-induced contraction in the presence of nimodipine, suggesting that pinacidil has actions other than the opening of potassium channels and subsequent membrane hyperpolarization. Bay K 8644 increased the NA potency fourfold in the presence of rauwolscine, whereas it had no effect on the NA response in the presence of prazosin and in the absence of alpha-adrenoceptor blockade. Such an action of Bay K 8644 can be reconciled with alpha 1-adrenoceptor activation causing membrane depolarization and opening of potential-operated calcium channels. It may be concluded that both alpha 1- and alpha 2-adrenoceptor-mediated contractions in human hand veins are highly dependent on Ca2+ influx, although the mechanisms utilized to bring about this influx partly differ between the two receptor subtypes.     

Pharmacological properties of prejunctional α-adrenoceptors in isolated feline middle cerebral arteries; comparison with the postjunctional α-adrenoceptors

In cat middle cerebral arteries (CMCA) preincubated with ³H-noradrenaline (NA), the outflow of tritium evoked by electrical stimulation was reduced to 32% by α-adrenoceptor (α-receptor) stimulation with oxymetazoline, and increased to 487% by α-receptor blockade with HEAT. The relative order of potency for α-receptor agonists on prejunctional receptors was: clonidine ≥ oxymetazoline > phenylephrine, and the antagonist rauwolscine was more potent than prazosin. This indicates that the prejunctional α-receptors are mainly of α₂-type. Rauwolscine was more potent than prazosin in inhibiting the contractions induced by NA, indicating a predominance of α₂-receptors postjunctionally. Apart from clonidine having higher intrinsic activity pre- than postjunctionally, all drugs examined (oxymetazoline, phenylephrine, rauwolscine, HEAT (BE2254), and prazosin) had similar concentration-effect curves on the pre- and postjunctional receptors. Furthermore, the ratios of EC₅₀-values pre- and postjunctionally of rauwolscine, oxymetazoline, and clonidine were all close to unity. These results indicate that pre- and postjunctional α₂-receptors in the CMCA have similar pharmacological characteristics and cannot be influenced separately by the presently used drugs.     

Influence of extracellular calcium and nifedipine on α1-and α2-adrenoceptor-mediated contractile responses in isolated rat and cat cerebral and mesenteric arteries

The influence of extracellular Ca²⁺ and nifedipine on contractile responses to 10 μM noradrenaline (NA) was investigated in isolated rat and cat middle cerebral (RCA, CCA) and mesenteric (RMA, CMA) arteries. In the CCA (containing predominantly α₂-adrenoceptors), the NA-induced contractions developed considerably more slowly than in the RCA, RMA (containing mainly α₁-adrenoceptors) and CMA (sensitive to both at,- and α₂-adrenoceptor selective antagonists). The tonic component of the NA-induced contraction in the four types of artery was substantially suppressed after only short periods in Ca²⁺-free solution. In each type of artery, excluding the CCA, the contractile response to 124 mM K⁺ was more sensitive to Ca²⁺ deprivation than that to NA. This suggests that NA, besides mobilizing extracellular Ca²⁺, can also release Ca²⁺ from an intracellular pool in the RCA, RMA and CMA, but not in the CCA. Thus, α₁-adrenoceptor-mediated contractions in the RCA and RMA seem to depend on both Ca²⁺ influx and intracellular Ca²⁺ release, whereas α₂-adrenoceptor-mediated contractile responses in the CCA appear to rely almost entirely on Ca²⁺ influx. Both the maximum response and the tonic component of the NA-induced contraction were significantly more sensitive to nifedipine in the CCA than in the RCA. In comparison with the NA-induced contractions in these arteries, those in the RMA and CMA were relatively resistant to nifedipine. In the CCA exposed to NA in Ca²⁺-free medium, nifedipine almost abolished the contraction induced by re-addition of Ca²⁺, whereas in the other types of artery, Ca²⁺ re-application evoked a significant contraction also in the presence of the drug. The differential effects of nifedipine presumably reflect differences between the arteries, not only in the relative contribution of Ca²⁺ influx and intracellular Ca²⁺ release to the contractile activation, but also in the nifedipine sensitivity of the Ca²⁺ entry pathways utilized by NA. It is concluded that the mechanisms through which NA induces contraction seem to be related both to the subtype of α-adrenoceptor stimulated by NA and to the type of vessel studied.     

Postjunctional α1- and α2-adrenoceptors mediating contraction in isolated human groin arteries and veins

By means of selective agonists and antagonists for α₁- and α₂-receptors, the α-receptor subtypes in human groin arteries and veins were characterized and compared. In the arteries the α₁-receptor blocker prazosin caused a concentration-dependent parallel displacement of the noradrenaline (NA) concentration-response (cr) curve without reduction of maximum (pA₂=9.86); the selective α₂-receptor antagonist rauwolscine in the concentration 10^-8 M caused a right-ward shift of the NA cr-curve without reduction of E_max, but 10^-7 M and 10^-6 M caused little or no further shift. In the veins, the two antagonists had the opposite effects. Rauwolscine caused a concentration-dependent right-ward shift of the NA cr-curve without depression of maximum (pA₂=9.03); prazosin 10^-9 M significantly displaced the NA cr-curve, whereas 10^-8 M and 10^-7 M caused little or no further shift. The responses to the α₂-receptor agonist clonidine in the arteries were too small to allow calculations of pEC50 values; in the veins contractions were elicited in all vessel segments investigated (pEC₅₀=6.24). Phenylephrine, selective for α₁-receptors, was significantly more potent in arteries than in veins. NA was significantly more potent in veins than in arteries. It is concluded that in human groin vessels, there is a functional predominance of arreceptors in the arteries and of a2-receptors in the veins.     

Pharmacological characterization of postjunctional α-adrenoceptors in isolated human omental arteries and veins

The α-adrenoceptors in human omental arteries and veins were characterized and compared. In the arteries both prazosin (pA₂ 9.48) and rauwolscine (pA₂ 7.19) displaced the noradrenaline (NA) concentration-response (cr) curve towards higher concentrations without reduction of maximum. Neither clonidine, nor oxymetazoline had any consistent contractile effects. Phenylephrine had a lower potency than NA, but a similar intrinsic activity. In the veins, both prazosin (pA₂ 9.72) and rauwolscine (pA₂ 8.11) displaced the NA cr-curve towards higher concentrations, but also significantly depressed maximum. Clonidine and oxymetazoline contracted veins from 3 out of 7 and 4 out of 6 patients, respectively. Their pD₂-values were similar to that of NA, but their intrinsic activities were significantly lower. NA was more potent than phenylephrine in these vessels, and there was no significant difference in intrinsic activity. The results suggest that in human omental arteries, the postjunctional a-adrenoceptors are mainly of the α₁-type, even if a small population of α₂-adrenoceptors cannot be excluded. In omental veins, there seems to be a functionally important population of postjunctional α₂-adrenoceptors occurring together with a population of α₁-adrenoceptors.     

Selective α2-adrenoceptor activation by clonidine: effects on 45Ca2 efflux and insulin secretion from isolated rat islets

A possible role for Ca²⁺ in the α-adrenoceptor-induced inhibition of glucose-stimulated insulin secretion was studied in isolated rat islets by the use of the selective α₂-adrenoceptor agonist clonidine. We found that clonidine, in contrast to the a,-adrenoceptor agonist phenylephrine, inhibited glucose-stimulated insulin secretion at dose levels below 10^-6 mol l^-1. In islets preloaded with ⁴⁶Ca²⁺ and perifused at 2 mmol l ^l Ca²⁺, clonidine (10^-6 moll^-1) reduced the glucose (13.3 mmol l^-1)-stimulated ⁴⁶Ca²⁺efflux during both the first and second phases of insulin secretion. Furthermore, the inhibitory effect of clonidine on glucose (13.3 mmol l^-1)-stimulated insulin secretion was partially counteracted by raising the extracellular Ca²⁺ concentrations. Moreover, the calcium channel agonist Bay K 8644 counteracted the inhibition by clonidine on glucose-stimulated insulin secretion. Our results suggest that selective α₂-adrenoceptor-induced inhibition of glucose-stimulated insulin secretion is mediated, at least partially, by restraint of Ca²⁺-influx. This action might in turn be exerted through interference with the voltage-dependent calcium channels.     

Enhancement of insulin secretion during selective blockade of α1, - and α2-adrenoceptors in the rat: Effects of somatostatin

The effects of somatostatin on plasma concentrations of insulin and glucose in the presence of the selective α₁-adrenoceptor blocking agent prazosin or the selective α₂-adrenoceptor blocking drug yohimbine were studied in vivo in anesthetized rats. Infusion of both prazosin (0.080 mg/min) and yohimbine (0.018 mg/min) increased plasma insulin levels within 10 min. Prazosin, but not yohimbine, caused a significant increment in plasma glucose concentration. Somatostatin (0.1 μg/min) promptly and extensively lowered plasma insulin concentrations during the infusion of both prazosin and yohimbine, suggesting that the inhibitory effect of somatostatin is not mediated via a direct action on α₁ or α₂-adrenoceptors. Plasma glucose concentration fell slightly during somatostatin administration. A marked increment in insulin release occurred in response to cessation of the somatostatin infusion, both during prazosin- and yohimbine-treatment. We conclude that somatostatin efficiently inhibits insulin secretion during selective α₁- and α₂-adrenoceptor blockade and, further, that the insulin off-response after somatostatin treatment is potentiated by α-adrenoceptor blockade. This study also indicates that blockade of α₁- as well as of α₂-adrenoceptors leads to an increased insulin secretion.     

Influence of angiotensin II, α- and β-adrenoceptors on peripheral noradrenergic neurotransmission in canine gracilis muscle in vivo

Interactions between angiotensin II and adrenoceptor-mediated effects on peripheral sympathetic neurotransmission were investigated in constant flow blood-perfused canine gracilis muscle in situ, without and with pretreatment by non-competitive α- adrenoceptor blockade. Angiotensin converting enzyme (ACE)-inhibition by benazeprilat increased nerve stimulation (2 Hz, 4 min)-evoked noradrenaline (NA) overflow (+21 ± 5 yo) with α- adrenoceptors intact, but reduced NA overflow (– 18 ± 6%) when α-adrenoceptors were blocked. Vasoconstrictor responses were slightly reduced by benazeprilat. Subsequent infusion of angiotensin II (Ang 11, 20 and 500 ng kg_-1 min_-1 i.v., raising arterial concentrations from 0.6 ± 0.2 PM to 1390 ± 240 and 25 110 ± 3980 PM, respectively) failed to increase NA overflow or to enhance stimulation-evoked vasoconstriction. Adrenaline (0.4 nmol kg_-1 min_-1 i.v.) did not change evoked NA overflow before or after benazeprilat, either with or without α-adrenoceptor blockade, despite high concentrations (± 10 nM) in arterial plasma. Following benazeprilat, propranolol reduced NA overflow (–24 ± 3 yo) only if the α-adrenoceptors were blocked. In conclusion, benazeprilat reduced evoked NA overflow in the presence of α- adrenoceptor blockade to a similar degree as previously shown in the presence of neuronal uptake inhibition in this model. However, contrasting to our previous findings, benazeprilat enhanced NA overflow and reduced the post-junctional response to nerve stimulation in the absence of α-adrenoceptor blockade. This could be related to bradykinin accumulation during ACE-inhibition, in addition to the reduction of Ang II generation. Our data are not compatible with facilitation of NA release by circulating Ang II even at pharmacological dose levels. Although activation of prejunctional β-adrenoceptors may facilitate evoked NA overflow in this model, circulating adrenaline is ineffective under physiological conditions even after α-adrenoceptor blockade. Also, β-adrenoceptor-mediated prejunctional effects do not seem to involve Ang II in canine skeletal muscle in vivo.     

Pharmacological characterization of postjunctional α-adrenoceptors in isolated feline cerebral and peripheral arteries

The vascular α-adrenoceptors in isolated feline cerebral, lingual and mesenteric arteries were characterized and compared. In the middle cerebral artery the relative order of potency for agonists was: clonidine>oxymetazoline>noradrenaline>phenylephrine which indicates that the postjunctional α-receptor in this vessel is of α₂-type. This view is further supported by the finding that yohimbine, but not prazosin, had a potent, mainly competitive blocking action. In peripheral arteries, clonidine was without effect. In these vessels, the potency difference between phenylephrine and oxymetazoline was more than 40 times less than in cerebral vessels. The pA₂-values for prazosin correlated well with pA₂-values found for the interaction of this drug with α₁-receptors in a variety of other tissues, thus suggesting the occurrence of an α₁-receptor in these arteries. However, the pA₂-values for yohimbine and rauwolscine correlated well with an α₂-receptor, suggesting also the presence of α₂-receptors. Schild plots for prazosin and rauwolscine in lingual arteries displayed slopes significantly lower than unity, which also supports the view of a mixture of α₁-and α₂-receptors in these vessels. However, the Schild plots for the antagonists in mesenteric arteries did not differ significantly from unity, a finding possibly indicating the presence of an α-receptor unable to differentiate between substances that in other tissues act preferentially on α₁- or α₂-receptors.     

Prostaglandin E2- and α- or β-Adrenoceptor Mediated Interferences with 3H-Noradrenaline Secretion from Human Vasomotor Nerves: Comparison between Effects on Omental Arteries and Veins

In earlier work with human vasomotor nerves (Stjärne and Gripe 1973) it has been shown that the inhibition of ³H-NA secretion by exogenous N A, and the enhancement caused by the α-adrenoceptor antagonist phentolamine, occur even after blocking formation of prostaglandin E₂ (PGE₂) with 5,8,11,14-eicosatetraynoic acid (ETA). This shows that at least part of the α-adrenoceptor mediated control of NA secretion is independent of (not mediated by) endogenous PGE₂. In the same paper it was also shown that the inhibitory effect of exogenous PGE₂ on ³H-NA secretion persists in the presence of phentolamine, indicating that exogenous PGE₂ acts on a target in the nerves different from that of NA (Stjärne 1973).     

α2-Aclrenoceptor agonist-mediated inhibition of [3H]noradrenaline release from rat hippocampus is reduced by 4-aminopyridine,but that caused by an adenosine analogue or co-conotoxin is not

The inhibitory effect of an adenosine analogue, R-PIA, and an α₂-adrenoceptor agonist, UK 14,304, on [³H]NA efflux from field-stimulated rat hippocampal slices was examined. The effect of 0.1 μm UK 14,304 was mimicked by 30 nM w-conotoxin and by 10 μM cadmium chloride, inhibitors of N- and L-type Ca²⁺ channels. R-PIA (1 μM) had no effect per se, but caused a clear-cut inhibition after blockade of the pre-synaptic α₂-receptor by yohimbine. 4-Aminopyridine (4-AP) caused a dose-dependent increase in evoked transmitter release. At 30 μM 4-AP did not affect the actions of ω-conotoxin or cadmium chloride. The pre-synaptic effect of R-PIA was similarly unaffected by 30 μM 4-AP. The presynaptic effect of UK 14,304 was virtually abolished by 4-AP (30 μM). The effect of UK 14,304 (0.1 μM) could be partly restored by reducing the Ca²⁺ concentration during treatment with 4-AP (22% inhibition compared to 42% with normal Ca²⁺). The magnitude of increase in evoked [³H]NA efflux by yohimbine (1 μM) was decreased by 4-AP in a concentration-dependent manner from 142% increase in controls to 21 % at 100 μM 4-AP. The present results indicate that NA release is reduced by somewhat different mechanisms by pre-synaptic α₂- and adenosine Aj-receptors. Furthermore, the results indicate that pre-synaptic A_rreceptors on hippocampal NA neurons do not primarily regulate 4-AP-dependent potassium channels, but they might act directly on a Ca²⁺ conductance.     

Effects of selective α1 and α2 adrenoceptor active drugs on 86Rb+ efflux from pieces of rat parotid gland

Minute pieces of rat parotid gland were used in studies of adrenergic regulation of K+ efflux using ⁸⁶Rb+ as a probe for K+. Noradrenaline induced a concentration-dependent Rb+ efflux, whereas the β₁-selective agonist prenalterol was without effect. On the other hand, the β₂-selective drug, terbutaline, at high concentrations displayed a small enhancement of Rb+ -secretion. The selective α₁-adrenoceptor drug, phenylephrine, was as potent as noradrenaline, whereas the α₂-agonist clonidine had only a small effect. The noradrenaline-induced Rb+-efflux was effectively inhibited in the presence of prazosin, an α₁-blocker, whereas the α₂-antagonist, yohimbine, was roughly 50 times less potent. The results suggest that catecholamine-induced K+-secretion from the rat parotid gland is mediated via activation of post-synaptic α-adrenoceptors of the α₁-subtype.     

Reversal of α1-receptor mediated relaxation in intestinal smooth muscle

The α₁-receptor agonist phenylephrine relaxed longitudinal rabbit jejunal muscle contracted in vitro by low concentrations of barium ions (1 mM). When the Ba²⁺ concentration was increased to 10–15 mM the response to phenylephrine was a contraction, and at Ba²⁺ concentrations in between the high and low range this response was biphasic—a relaxation followed by a contractile phase. The α₂-receptor agonist clonidine did not affect the tone of the Ba²⁺ contracted preparation. When the muscle preparation was contracted by Sr²⁺ (1–20 mM) in the presence of Ca²⁺ (2.5 mM), phenylephrine relaxed it, and no contractile response to phenylephrine was observed. In the absence of extracellular Ca²⁺, 5 mM Ba²⁺ caused a contraction. Under these conditions phenylephrine had no effect on the tissue tone. When Ca²⁺ was added in a low concentration (0.2-2 mM), phenylephrine elicited a gradually increasing contractile response. At 5 mM Ca²⁺ the contractile response was replaced by the normal relaxation. The contractile response to phenylephrine in the presence of 5 mM Ba²⁺ and 2.5 mM Ca²⁺ was partially blocked by low concentrations of verapamil. In higher concentrations verapamil abolished the tissue tonus completely. The contractile response to phenylephrine in the presence of 5 mM Ba²⁺ and 2.5 mM Ca²⁺ could be reverted to the normal relaxation by the addition of 20 mM Mg²⁺. Increasing the K⁺ concentration from the normal 5.9 to 62.9 mM blocked the phenylephrine-induced relaxation. No contractile response to phenylephrine occurred. It is concluded that Ba²⁺ could reverse the response of α₁ receptor stimulation in rabbit jejunum from a relaxation to a contraction and that this contractile response was dependent on the presence of Ca²⁺.     

Effects of some calcium antagonists on aggregation by adrenalin and serotonin and on α-adrenoceptor radioligand binding in human platelets

The inhibitory effects of verapamil, nifedipine and diltiazem, representatives of different classes of calcium antagonists, were studied on aggregation of human platelets induced by adrenalin and serotonin (5-HT). For references, the α-adrenoceptor-antagonists phentolamine (α₁ and α₂) and rauwolscine (α₂), and the s-HT₂-receptor-antagonist ketanserin were included. Verapamil in the concentration range 10^-6- 10^-4 M inhibited both adrenalin- and serotonin-induced aggregation in a concentration-dependent manner, whereas nifedipine and diltiazem had little or no effect. Phentolamine and rauwolscine were clearly weaker than verapamil as antagonists of serotonin, and ketanserin lacked effect on adrenalin-induced aggregation. Binding studies with [³H]dihydro-α-ergocryptine and [³H]rauwolscine on human platelet membranes showed equal numbers of binding sites, suggesting that only α₂-adrenoceptors were present. In the same concentration range as inhibition of aggregation was obtained, verapamil inhibited binding of either radioligand. Nifedipine, diltiazem and 5-HT were all poor inhibitors of radioligand binding. The results suggest that verapamil at high concentrations not only has α-adrenoceptor antagonistic properties but also exerts 5-HT-receptor blocking effects. This was not found with the other calcium channel blockers examined (nifedipine, diltiazem).     

Effects of α- and β-adrenoceptor stimulation on 45Ca2+ efflux and insulin secretion from perfused rat islets

Exposure to the β₂-adrenoceptor agonist terbutaline resulted in a transient stimulation of ⁴⁵Ca²⁺ efflux from ⁴⁵Ca²⁺ preloaded rat islets perfused in 2 mm Ca²⁺ and 8.3 mm glucose. Concomitantly, an increase in insulin secretion occurred. Under the same experimental conditions, the α-adrenoceptor agonist noradrenaline promptly inhibited insulin release without any apparent influence on ⁴⁵Ca²⁺ efflux. In contrast, in a medium containing 2 mm Ca²⁺ and a low glucose concentration (2.8 mm), terbutaline stimulated insulin secretion without any apparent effects on ⁴⁵Ca²⁺ efflux. Noradrenaline had no effect on insulin secretion or ⁴⁵Ca²⁺ efflux in this medium. When islets were perfused with 8.3 mm glucose in a Ca²⁺ deficient medium, with or without addition of the chelating agent EGTA, terbutaline induced a marginal stimulation of insulin secretion and a negligible stimulation of ⁴⁵Ca²⁺ efflux. On the contrary, noradrenaline stimulated to an immediate and notable ⁴⁵Ca²⁺ efflux in these Ca²⁺ deficient media. Noradrenaline also clearly inhibited insulin secretion, though less markedly and with a slower onset than in islets perfused in 2 mm Ca²⁺. When the islets were perfused in a Ca²⁺ deficient medium with 2.8 mm glucose, terbutaline had a slight insulin releasing effect, but stimulated ⁴⁵Ca²⁺ efflux potently. Noradrenaline had no influence on insulin secretion but a weak stimulatory effect on ⁴⁵Ca²⁺ efflux. The data suggest that the β₂-adrenoceptor agonist terbutaline has the ability to stimulate insulin secretion in perfused rat islets, requiring extracellular Ca²⁺ for the full expression of its effects. These effects may be exerted through a Ca²⁺-Ca²⁺ exchange over the cell membrane and/or through cAMP and intracellular Ca²⁺ perturbations. Moreover, terbutaline directly stimulates ⁴⁵Ca²⁺ efflux, an effect inhibited by glucose. Further, the α-adrenoceptor agonist noradrenaline can inhibit insulin secretion in the absence of extracellular Ca²⁺, but the full expression of its inhibitory action is dependent on extracellular Ca²⁺ and glucose. In addition, noradrenaline stimulates ⁴⁵Ca²⁺ efflux in a Ca²⁺ deficient medium, an effect which appears independent of the glucose concentration.     

Decreased responsiveness of vascular postjunctional α1-, α2-adrenoceptors and neuropeptide Y1 receptors in rats with heart failure

Heart failure is associated with increased sympathetic nerve activity. We hypothesized that chronic sympathetic stimulation in heart failure resulted in decreased vascular sympathetic responsiveness. A pithed rat model was employed to evaluate peripheral vascular α-adrenoceptor and neuropeptide Y (NPY) receptor responsiveness. Heart failure was induced in Sprague–Dawley rats by coronary artery ligation. Sham operated rats (Sham) served as controls. Two months after this surgical procedure, both heart failure (n= 30) and Sham (n= 30) rats underwent standard pithing procedure. Pressor responses to preganglionic sympathetic nerve stimulation (PNS) and activation of postjunctional α₁- and α₂-adrenoceptors as well as Y1 receptors were studied. In response to PNS, cardiac index was similar between heart failure and sham rats (P= n.s.). Mean arterial pressure (MAP) increased in a frequency-dependent fashion after PNS in heart failure rats as well as in control rats. All the agonists used, i.e. the α₁-adrenoceptor agonist phenylephrine, the α₂-adrenoceptor agonists clonidine and BHT933 as well as NPY, induced dose-dependent increases in MAP in heart failure and in sham rats. However, in rats with heart failure, the response to all the agonists studied was significantly decreased and the dose response curves were shifted to the right (P < 0.01). We conclude that in vivo vascular response to postjunctional α₁- and α₂-adrenoceptors as well as Y1 receptors are decreased in rats with heart failure.     

High spatial resolution studies of muscarinic neuroeffector junctions in mouse isolated vas deferens

It is acknowledged that neurotransmission in the mouse vas deferens is predominantly mediated by ATP and noradrenaline (NA) released from sympathetic nerves while cholinergic transmission in the rodent vas deferens is often overlooked despite early literature. Recently we have characterized a cholinergic component of neurogenic contraction of mouse isolated vas deferens. In the present paper, by confocal imaging of Ca²⁺ dynamics we detected acetylcholine (ACh) action at muscarinic cholinergic neuroeffector junctions at high-resolution. Experiments were carried out in the presence of prazosin (100 nM) and α,β methylene ATP (α,β-MeATP) (1 μM) to inhibit responses to NA and ATP respectively. Exogenous ACh (10 μM) elicited Ca²⁺ transients, an effect blocked by the muscarinic receptor antagonist, cyclopentolate (1 μM). Ca²⁺ transients were evoked by electrical stimulation of intrinsic nerves in the presence of the cholinesterase inhibitor neostigmine (10 μM). Stimulation produced a marked increase in the frequency and number of Ca²⁺ transients. Cyclopentolate reduced the frequency of occurrence of spontaneous and evoked events to control levels. The α₂-adrenoceptor antagonist yohimbine (300 nM) did not affect the spontaneous Ca²⁺ transients, but increased the frequency of occurrence of evoked transients, an effect inhibited by cyclopentolate. The postjunctional effects of neuronally-released ACh are limited by the action of cholinesterase. Release of ACh appears to be tonically inhibited by NA released from sympathetic nerve terminals through action at prejunctional α₂-adrenoceptors. Tetrodotoxin (TTX, 300 nM) abolished the nerve-evoked Ca²⁺ events, with no effect on Ca²⁺ transients elicited by exogenous ACh. In conclusion, the presence of spontaneous and evoked cholinergic Ca²⁺ transients in smooth muscle cells of the mouse isolated vas deferens has been revealed. These events are mediated by ACh acting at M₃ muscarinic receptors. This action stands in marked contrast to the lack of effect of neuronally-released NA on smooth muscle Ca²⁺ dynamics in this tissue.     

Affinity of Noradrenaline and Dopamine for Neural α-Receptors Mediating Negative Feedback Control of Noradrenaline Secretion in Human Vasoconstrictor Nerves

Isolated superfused field stimulated biopsy specimens of human peripheral arteries and veins, preincubated with ³H-(-)-noradrenaline (NA) to label the neural stores of NA, were used to study the potency of dopamine (DA) and of NA as triggers of α-adrenoceptor mediated negative feedback control of sympathetic neurotransmitter secretion, evoked by stimulation with trains of 300 shocks at 1 Hz. In this preparation DA was found to be only slightly less potent than NA in depressing both the secretion of ³H-NA, and the contractile response, evoked by nerve stimulation. DA depressed the contraction evoked by exogenous NA as well, but to a very much smaller extent. On the other hand, DA was a very weak agonist on the α-receptors of the smooth muscle; nearly 1 000 times higher concentrations of DA were required to mimick contractions evoked by exogenous NA. The results show that the neural α-receptor function involved in control of NA secretion differs considerably from the α-receptors of e.g. smooth muscle, with respect to sensitivity to DA. It seems possible that the observed depressing effect of DA on NA secretion may be of pharmacological and clinical interest; it may at least in part explain the vasodilating effect of DA infusions in man.     

α 2-Adrenoceptors activate dihydropyridine-sensitive calcium channels via Gi-proteins and protein kinase C in rat portal vein myocytes

The presence of functional ₂-adrenoceptors was investigated in isolated smooth muscle cells from rat portal vein using the nystatin-perforated patch-clamp technique. The free cytoplasmic calcium concentration ([Ca²⁺]_i) was estimated using emission from the dye Fura-2. Activation of ₂-adrenoceptors by clonidine (an ₂-adrenoceptor agonist) or noradrenaline (a non-selective -adrenoceptor agonist), both in the presence of 0.1 M prazosin to block ₁-adrenoceptors, caused a slow and sustained increase in [Ca²⁺]_i which was inhibited by 0.1 M rauwolscine (an ₂-adrenoceptor antagonist). A similar Ca²⁺ response was obtained with oxymetazoline (a selective _2A-adrenoceptor agonist) suggesting that the increase in [Ca²⁺]_i resulted from activation of the _2A-adrenoceptor subtype. The increase in [Ca²⁺]_i did not occur in calcium-free solution or in the presence of oxodipine (a voltage-dependent calcium channel blocker), indicating that it depended on a calcium influx. The _2A-adrenoceptor-activated calcium influx was unchanged after complete release of the stored calcium induced by applications of ryanodine and caffeine. In addition, no accumulation of inositol trisphosphate was detected in the presence of 0.1 M prazosin. Taken together, these results indicate that _2A-adrenoceptor activation does not stimulate phosphoinositide turnover and subsequent calcium release from intracellular stores. Wholecell patch-clamp experiments showed that _2A-adrenoceptor activation promoted calcium influx through voltage-dependent L-type channels. Concomitant with calcium influx, _2A-adrenoceptor activation induced a 10- to 15-mV depolarization. Similar effects on both calcium channel current and [Ca²⁺]_i were obtained with mastoparan, an activator of Gi-proteins. Activation of calcium influx by both _2A-adrenoceptors and mastoparan was reduced by treatment with pertussis toxin and GF 109203X (a protein kinase C inhibitor). These data suggest that activation of protein kinase C through a transduction pathway involving Gi-proteins phosphorylates voltage-activated L-type calcium channels and thus, increases their opening probability.     

Dihydropyridine block of voltage-dependent K currents in rat dorsal root ganglion neurons

The dihydropyridines nifedipine, nimodipine and Bay K 8644 are widely used as pharmacological tools to assess the contribution of L-type voltage-gated Ca²⁺ channels to a variety of neuronal processes including synaptic transmission, excitability and second messenger signaling. These compounds are still used in neuronal preparations despite evidence from cardiac tissue and heterologous expression systems that they block several voltage-dependent K⁺ (Kv) channels. Both because these compounds have been used to assess the relative contribution of L-type Ca²⁺ channels to several different processes in dorsal root ganglion (DRG) neurons and because a relatively wide variety of Kv channels present in other neuronal populations is present in DRG neurons, we determined the extent to which dihydropyridines block Kv currents in these neurons. Standard whole cell patch clamp techniques were used to study acutely disassociated adult rat DRG neurons. All three dihydropyridines tested blocked Kv currents in DRG neurons; IC₅₀ values (concentration resulting in an inhibition that is 50% of maximum) for nifedipine and nimodipine-induced block of sustained Kv currents were 14.5 and 6.6 μM, respectively. The magnitude of sustained current block was 44±1.6%, 60±2%, and 56±2.9% with 10 μM nifedipine, nimodipine and Bay K 8644, respectively. Current block was occluded by neither 4-aminopyridine (5 mM) nor tetraethylammonium (135 mM). Dihydropyridine-induced block of Kv currents was not associated with a shift in the voltage-dependence of current activation or inactivation, the recovery from inactivation, or voltage dependent block. However, there was a small use-dependence to the dihydropyridine-induced block. Our results suggest that several types of Kv channels in DRG neurons are blocked by mechanisms distinct from those underlying block of Kv channels in cardiac myocytes. Importantly, our results suggest that if investigators wish to explore the contribution of L-type Ca²⁺ channels to neuronal function, they should consider alternative strategies for the manipulation of these channels than the use of dihydropyridines.