Actions of 4-chloro-3-ethyl phenol on internal Ca2+ stores in vascular smooth muscle and endothelial cells.

1. Recently, 4-chloro-3-ethyl phenol (CEP) has been shown to cause the release of internally stored Ca2+ apparently through ryanodine-sensitive Ca2+ channels, in fractionated skeletal muscle terminal cisternae and in a variety of non-excitable cell types. Its action on smooth muscle is unknown. In this study, we characterized the actions of CEP on vascular contraction in endothelium-denuded dog mesenteric artery. We also determined its ability to release Ca2+, by use of Ca2+ imaging techniques, on dog isolated mesenteric artery smooth muscle cells and on bovine cultured pulmonary artery endothelial cells. 2. After phenylephrine-(PE, 10 microM) sensitive Ca2+ stores were depleted by maximal PE stimulation in Ca2+-free medium, the action of CEP on refilling of the emptied PE stores was tested, by first pre-incubating the endothelium-denuded artery in CEP for 15 min before Ca2+ was restored for a 30 min refilling period. At the end of this period, Ca2+ and CEP were removed, and the arterial ring was tested again with PE to assess the degree of refilling of the internal Ca2+ store. 3. In a concentration-dependent manner (30, 100 and 300 microM), CEP significantly reduced the size of the post-refilling PE contraction (49.4, 28.9 and 5.7% of control, respectively) in Ca2+-free media. This suggests that Ca2+ levels are reduced in the internal stores by CEP treatment. CEP alone did not cause any contraction either in Ca2+-containing or Ca2+-free Krebs solution. 4. Restoring Ca2+ in the presence of PE caused a large contraction, which reflects PE-induced influx of extracellular Ca2+. The contraction of tissues pretreated with 300 microM CEP was significantly less compared with controls. However, tissues pretreated with 30 and 100 microM CEP were unaffected. Washout of CEP over 30 min produced complete recovery of responses to PE in Ca2+-free and Ca2+-containing medium suggesting a rapid reversal of CEP effects. 5. Concentration-response curves were constructed for PE, 5-hydroxytryptamine (5-HT) and K+ in the absence of and after 30 min pre-incubation with 30, 100 and 300 microM CEP. In all cases, CEP caused a concentration-dependent depression of the maximum response to PE (84.8, 43.4 and 11.6% of control), 5-HT (65.4, 25.7 and 6.9% of control) and K+ (77.6, 41.1 and 10.8% of control). 6. Some arterial rings were pre-incubated with ryanodine (30 microM) for 30 min before the construction of PE concentration-response curves. In Ca2+-free Krebs solution, ryanodine alone did not cause any contraction. However, 58% (11 out of 19) of the tissues tested with ryanodine developed contraction (6.9+/-1.2% of 100 mM K+ contraction, n=11) in the presence of external Ca2+. EC50 values for PE in ryanodine-treated tissues (1.7+/-0.25 microM, n=16) were not significantly different from controls (2.5+/-0.41 microM, n=22). Maximum contractions to PE (118.5+/-4.4% of 100 mM K+ contraction, n=16) were also unaffected by ryanodine when compared to controls (129+/-4.2%, n=23). 7. When fura-2 loaded smooth muscle cells (n=13) and endothelial cells (n=27) were imaged for Ca2+ distribution, it was observed that 100 and 300 microM CEP in Ca2+-free medium caused Ca2+ release in both cell types. Smooth muscle cells showed a small decrease in cell length. Addition of EGTA (5 mM) reversed the effect of CEP on intracellular Ca2+ to control values. 8. These data show, for the first time in vascular smooth muscle and endothelial cells, that CEP releases Ca2+ more rapidly than ryanodine. Unlike ryanodine, CEP caused no basal contraction but depressed contractions to PE, 5-HT and K+. The lack of basal contraction may result from altered responsiveness of the contractile system to intracellular Ca2+ elevation.

Interactions of chloroethylclonidine with rauwolscine- and prazosin-sensitive adrenoceptors in dog saphenous vein.

1. alpha 1-Adrenoceptors have been classified pharmacologically into four subtypes (alpha 1A, alpha 1B, alpha 1C and alpha 1D) on the basis of their differential affinity for novel antagonists such as chloroethylclonidine (CEC). While CEC is considered an alpha 1B-adrenoceptor antagonist, our earlier studies revealed that it also acted like an agonist in the dog saphenous vein (DSV). The present study characterized the contraction induced by CEC in endothelium-denuded rings from DSV. 2. Concentration-response curves for CEC were constructed in the absence (EC50 value of 11.13 +/- 3.6 microM, n = 8) and presence of propranolol (beta-adrenoceptor antagonist, 30 nM), rauwolscine (alpha 2-adrenoceptor antagonist, 30 nM), prazosin (alpha 1-adrenoceptor antagonist, 30 nM) or methysergide (5HT2 antagonist, 30 nM) or both prazosin and rauwolscine. Pretreatment with methysergide (9.83 +/- 5.14 microM, n = 4) or propranolol (23.78 +/- 12.32 microM, n = 4) had no consistent effect. In the presence of rauwolscine, the concentration-response curve for CEC was significantly shifted to the right with an EC50 value of 48.82 +/- 13.2 microM (n = 8). In the presence of prazosin, the CEC concentration-response curve had an EC50 value of 29.12 +/- 6.42 microM (n = 8). Pretreatment with both prazosin and rauwolscine shifted the concentration-response curve for CEC to the right with an EC50 value of 72.67 +/- 10.69 microM (n = 8, P < 0.05). Maximum responses were significantly reduced only in tissues that were treated with both prazosin and rauwolscine. 3. CEC (100 microM) pretreatment abolished prazosin binding sites and reduced the Bmax for rauwolscine by 50% without affecting the Kd value or the Hill slope.4. In Ca2+-free Krebs solution containing 50 microM EGTA, CEC produced a small transient contraction,suggesting that it can mobilize internally-stored Ca2+ . Pretreatment with rauwolscine abolished the CEC-induced contraction in Ca2+-free medium; prazosin pretreatment reduced but did not abolish CEC response in Ca2+-free medium.5. Restoring Ca2+ (0.5-2.5 mM) to the extracellular solution increased CEC contraction in a concentration-dependent manner, reaching a plateau at around 1.5mM Ca2 . The contraction was insensitive to nicardipine (1 microM), a voltage-operated Ca2+ channel blocker, but was blocked in a concentration-dependent manner by the putative receptor-operated Ca2+ channel blockers, SK&F 96365(1-1O microM) and genistein, also a tyrosine kinase inhibitor (10-100 microM).6. We conclude that CEC acts on rauwolscine- and, to a less extent, prazosin-sensitive adrenoceptors inDSV to release internally stored Ca2+ and to open receptor-operated Ca2+ channels. The inhibitory effect on CEC-induced contraction that depended on external Ca2+ by genistein suggests a role forty rosine kinase in the regulation of dihydropyridine-insensitive Ca2+ entry.

Sensitivity to protein kinase C inhibitors of nicardipine-insensitive component of high K+ contracture in rat and guinea-pig aorta.

1. In the rat and guinea-pig aorta, we observed that the contraction to hypertonically-added K+, unlike the isotonic K(+)-induced contraction, was only partially sensitive to nicardipine (0.1, 1 and 10 microM), an L-type Ca2+ channel blocker and occurred in Ca(2+)-free medium containing 50 microM EGTA. We have characterized this nicardipine-insensitive hypertonically-added K+ contraction. 2. The contraction induced by an equi-osmolar concentration of mannitol was similar in size to that evoked by hypertonically-added K+. 3. When the tissue was depleted of its internal Ca2+ stores with various agents such as phenylephrine (10 microM) cyclopiazonic acid (30 microM), thapsigargin (1 microM) or ryanodine (30 microM), or by incubation in Ca(2+)-free medium over 30 min, little effect was observed on the high K+ contracture in the presence of L-type Ca2+ channel blockade. 4. Phentolamine (10 microM) or indomethacin (10 microM) did not reduce the contraction induced by high K+. 5. Application of a protein kinase C inhibitor, H7 (10, 30 and 100 microM) or calphostin C (1 microM), reduced the high K+ contraction but not that caused by an equi-osmolar concentration of mannitol. 6. The data suggest that hypertonic K(+)-induced contraction differs from that caused by hypertonicity or depolarization per se and invokes membrane enzyme activation.

Unusual alpha-adrenoceptor subtype in canine saphenous vein: comparison to mesenteric vein.

1. We investigated the nature of the adrenoceptors in the dog saphenous vein (DSV) and dog mesenteric vein (DMV) to determine the nature of the unexpected interactions of phenylephrine and methoxamine with rauwolscine in the DSV, i.e. the ability of the putative alpha 2-adrenoceptor antagonist to inhibit competitively contractions to these alpha 1-agonists. Radioligand binding studies were performed in parallel with contractility studies. 2. Functionally, in the DSV, phenylephrine and methoxamine-induced, contractions were antagonized by rauwolscine with Schild slopes of -0.52 and -0.46, respectively and apparent pA2 values of 8.5 and 9.2, respectively. Such antagonism was not observed in the DMV. In the DSV, prazosin competes for [3H]-rauwolscine binding sites with a high and a low affinity binding site (Ki of 1.49 +/- 0.65 and 94.7 +/- 51 microM, n = 6, respectively). 3. Pretreatment with 100 microM chloroethylclonidine (CEC) for 15 min abolished [3H]-prazosin binding in microsomes from both veins and reduced binding (Bmax) of [3H]-rauwolscine in microsomes by 55.1 +/- 0.8% (n = 3) in the DSV but did not affect the Bmax in the DMV. CEC pretreatment in the venular rings denuded of endothelium caused persistent contraction in the DSV but not in the DMV. In the DSV, CEC appeared to interact with a single [3H]-rauwolscine binding site. In both the DSV and the DMV, CEC (100 microM) caused a significant shift in the EC50 values for phenylephrine and methoxamine. Maximum responses in the DMV were significantly attenuated while those in the DSV were unaffected when total tension was considered. 4. Studies of the functional interactions of the DSV and the DMV with WB 4101 or 5-methylurapidil (5-MU) suggested the presence of alpha 1D-adrenoceptors in the DSV and alpha 1A-adrenoceptors in the DMV. The receptors inactivated by CEC in the DMV and DSV may represent some or all of the receptors with properties of alpha 1D and alpha 1A-receptors present in the two veins. Studies of radioligand binding interactions of these two antagonists with [3H]-prazosin, were consistent with the presence of some alpha 1D-receptors in DSV and alpha 1A-receptors in DMV. These findings raise questions about the selectivity of CEC in differentiating alpha 1-adrenoceptor subtypes. 5. B-HT 920 caused contractions in the DSV smaller than those to the alpha 1-agonists but the maximum was not affected by CEC pretreatment. The EC50 values were shifted to the left after CEC. In radioligand binding studies, B-HT 920 competition for [3H]-rauwolscine binding was not significantly affected by CEC pretreatment. 6. These results suggest the presence of unusual alpha-adrenoceptors in the DSV. In addition to alpha 2-adrenoceptors, receptors recognizing rauwolscine as well as prazosin, WB 4101, phenylephrine and methoxamine and susceptible to inactivation by CEC are present. They appear to be, in part, unusual alpha 1D-adrenoceptors.

Mobilization of internal Ca2+ by vasoactive intestinal polypeptide in endothelial cells.

The aims of the present study were to establish whether vasoactive intestinal polypeptide (VIP) could mobilize internally-stored Ca2+ and whether Ca2+ release could trigger Ca2+ influx from the extracellular space. Bovine pulmonary artery endothelial cells from an established cell line were loaded with fura-2/AM and cells were studied in suspension or were imaged in monolayers at 40-80% confluency. In Ca2+ imaging studies, VIP evoked Ca2+ transients in Ca2+-free medium containing 50 microM EGTA. This was observed in 33 out of 122 cells examined on 29 separate trials. With each cell, the spread of Ca2+ appeared to occur from the periphery of the cell to the central core. Cells which did not respond to VIP responded to other stimulants such as bradykinin, endoplasmic reticulum Ca2+ pump inhibitors, (cyclopiazonic acid and thapsigargin), and endoplasmic reticulum Ca2+ release channel opener, ryanodine. The reintroduction of Ca2+ following VIP-induced Ca2+ release did not evoke a Ca2+ response in 5 cells imaged. Cells in suspension showed typical biphasic responses to bradykinin, thapsigargin or cyclopiazonic acid in the presence of external Ca2+. Stimulation with VIP caused transient Ca2+ responses in Ca2+-free physiological saline containing 50 microM EGTA. However, only 1 out of 4 cells tested showed a response to Ca2+ when it was reintroduced to the bathing medium. This study provided direct evidence for the first time in these bovine endothelial cells for VIP-mediated elevation of cytosolic concentration of Ca2+. The results also suggested that other mechanisms might prevail preventing capacitative Ca2+ entry following the release of internally-stored Ca2+.

Effects of thapsigargin and ryanodine on vascular contractility: cross-talk between sarcoplasmic reticulum and plasmalemma.

Thapsigargin and ryanodine are proposed to interfere with Ca2+ storage in sarcoplasmic reticulum by different mechanisms. Thapsigargin inhibits Ca2+ transport into and ryanodine enhances Ca2+ out of the sarcoplasmic reticulum. Contractility studies were performed in the rat aorta and dog mesenteric artery. Ryanodine was found to reduce phenylephrine-induced (10 microM) contraction in Ca(2+)-free medium of rat aorta and dog mesenteric artery in a concentration-dependent manner. Each agent alone caused a slow contraction in the rat aorta. In this tissue, the tension caused by ryanodine (30 microM) but not that by thapsigargin (1 microM) was found to be dependent on the status of the sarcoplasmic reticulum: prior stimulation with K+ (60 mM) enhanced the rate of development of ryanodine-induced tension compared with when the sarcoplasmic reticulum was previously depleted with phenylephrine stimulation in Ca(2+)-free medium. Sodium nitroprusside (1 microM) or isoproterenol (1 microM) fully antagonized the contraction induced by ryanodine or phenylephrine. However, thapsigargin-induced contraction was antagonized fully by sodium nitroprusside and only partially by isoproterenol. This result suggests that cAMP elevation by isoproterenol required a functioning sarcoplasmic reticulum Ca2+ pump for its relaxant effect while cGMP elevation by sodium nitroprusside did not. These findings are consistent with the view that ryanodine promotes Ca2+ release from the sarcoplasmic reticulum and that thapsigargin inhibits the ability of cAMP to stimulate Ca2+ uptake into the store by blocking its Ca2+ pump. In the dog mesenteric artery, when the phenylephrine-sensitive Ca2+ pool was emptied and thapsigargin was added to block Ca2+ uptake into the store, restoration of Ca2+ in the Ca(2+)-free medium caused a transient contraction (absent in controls). This contraction was replaced by a significantly larger amplitude and more sustained contraction in low Na+ medium indicating the involvement of the Na+/Ca2+ exchanger in the homeostasis of cytosolic [Ca2+]. In the presence of nifedipine (2 microM), repletion of the phenylephrine-sensitive store was inhibited. It is possible that refilling occurs in part through L-type Ca2+ channels.

Plant alkaloids, tetrandrine and hernandezine, inhibit calcium-depletion stimulated calcium entry in human and bovine endothelial cells.

Depletion of internal Ca2+ stores causes capacitative Ca2+ entry which occurs through non-selective cation channels sensitive to blockade by SK&F 96365. Recently, alkaloids of Chinese herbal medicinal origin, tetrandrine and hernandezine, have been shown to possess actions including inhibition of Ca2+ channels in non-excitable cell types. In this study, we compared the actions of these novel inhibitors to those of SK&F 96365 in fura-2-loaded endothelial cells from human umbilical vein and bovine pulmonary artery. Depletion of Ca2+ from the internal stores was accomplished in Ca(2+)-free medium using an endoplasmic reticulum Ca2+ pump inhibitor, cyclopiazonic acid (CPA) or receptor agonists, histamine and bradykinin. Stimulation with histamine or bradykinin caused a marked and rapid transient increase in Ca2+ signal whereas CPA caused a smaller amplitude increase of longer duration. Restoring Ca2+ to the medium caused marked and sustained increases in the fluorescence indicating movement of Ca2+ into the cytosol presumably stimulated by the emptied Ca2+ stores. SK&F 96365 as well as tetrandrine and hernandezine antagonized depletion-induced Ca2+ entry. The results suggest that these putative inhibitors interact with Ca2+ entry triggered by depletion of the internal Ca2+ stores and their action is presumed to be on the non-selective cation channels. Their effectiveness may be enhanced by the mechanisms which lead to the opening of the Ca2+ influx channel.

The potential requirement for HIV counselling and voluntary testing in women of childbearing age.

This paper compares estimates of the potential HIV counselling and testing requirements in a genito-urinary medicine (GUM) clinic, where a formal HIV counselling service is provided, and in an antenatal clinic (ANC), where there is no formal HIV counselling service. Data were collected by means of questionnaires completed by women attending both clinics. Only 31% and 16% of counselling needs were being met at the GUM clinic and antenatal clinic respectively. At the GUM clinic 11% of respondents had had an HIV antibody test, and at the antenatal clinic 1% had been tested. In contrast 68% and 58% of respondents at the GUM and antenatal clinics respectively would accept the offer of an HIV antibody test. In low prevalence areas the universal offer of testing would greatly increase specialist counselling requirements, but alternative models of provision and selective testing may lead to a more efficient use of resources.

Role of tyrosine kinase on Ca2+ entry and refilling of agonist-sensitive Ca2+ stores in vascular smooth muscles.

Recent evidence suggests that some Ca2+ channels are regulated by a tyrosine kinase pathway. The possibility that Ca2+ entry is regulated by tyrosine kinase was investigated in canine vascular muscles. Functional studies were carried out in the dog mesenteric arteries and saphenous veins. Nicardipine-insensitive refilling of phenylephrine-sensitive Ca2+ stores of endothelium-denuded dog mesenteric artery, previously depleted of Ca2+, measured after a 30-min exposure of the arterial rings to Ca2+ in the absence and presence of genistein (10-100 microM), tyrphostin 23 (10-100 microM), tyrphostin 47 (10-100 microM), SK&F 96365 (3-30 microM), or vehicle, DMSO, as phenylephrine contraction in Ca(2+)-free medium, decreased with increasing concentrations of the inhibitors compared with DMSO-treated and control. Phenylephrine contraction in Ca(2+)-free medium was not affected in a consistent manner by tyrosine kinase inhibitors when added after the store refilling period, in Ca(2+)-free medium, 5 min prior to stimulation with phenylephrine, indicating that the inhibitors have no effect on the Ca2+ release mechanism or that they act directly on myofilaments. Cyclopiazonic acid (30 microM), an inhibitor of the sarcoplasmic reticulum Ca2+ pump, caused transient contractions in the dog saphenous vein only in the presence of extracellular Ca2+. These transient contractions were inhibited by tyrphostin 23 (30-100 microM), tyrphostin 47 (30-100 microM), genistein (30-100 microM), and SK&F 96365 (50-100 microM) but not by the vehicle DMSO. The results from this study provided evidence that a tyrosine kinase pathway is involved in the regulation of Ca2+ entry since inhibitors of tyrosine kinase are able to attenuate presumed Ca2+ entry following agonist-mediated Ca2+ store depletion. This route of Ca2+ entry is also sensitive to blockade by SK&F 96365.

Evidence for two types of internal Ca2+ stores in canine mesenteric artery with different refilling mechanisms.

Novel transient biphasic responses of the dog mesenteric artery to phenylephrine hydrochloride (PE, 10 microM) in Ca(2+)-free medium containing 50 microM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) have been analyzed. The initial component was significantly inhibited by ryanodine (30-100 microM), an agonist enhancing Ca2+ release from the sarcoplasmic reticulum, whereas the second was significantly inhibited by nifedipine (1 microM), and L-type Ca2+ channel antagonist, or EGTA, to chelate Ca2+, and was potentiated by BAY K 8644 (1 microM), an L-type Ca2+ channel agonist. After repletion of Ca2+ stores in normal Krebs solution or in high KCl (60 mM) Krebs, the first component was inhibited by cyclopiazonic acid (CPA, 30 microM), a putative, reversible, and selective microsomal Ca2+ pump adenosinetriphosphatase inhibitor. BAY K 8644 potentiated the second component in the presence of CPA. The inhibition of the first component by CPA suggests that the refilling ultimately requires the CPA-sensitive Ca2+ pump for Ca2+ resequestration. However, the second component may refill by a CPA-independent route opened by BAY K 8644. These results, taken as a whole, indicate that the biphasic PE response in Ca(2+)-free medium may reflect compartmentalization of Ca2+ storage related to the different routes of refilling.

Functional alterations in the aorta of the spontaneously hypertensive rat: pharmacological assessment with cyclopiazonic acid.

One of the changes in vascular smooth muscle membranes associated with hypertension is an alteration in Ca2+ handling. It has been unclear as to whether changes occurred at the plasma membrane or at the endoplasmic reticulum (ER) or both. Recently, cyclopiazonic acid (CPA) has been reported to inhibit selectively the ER Ca2+ pump. We aimed at determining the effect of ER Ca2+ pump inhibition by CPA on the contractility of aortic smooth muscle from 4- to 5-month-old SHR and age- and weight-matched WKY control rats. The responsiveness of the SHR tissues to phenylephrine or K+ was significantly reduced as compared with controls, although the sensitivity was not altered. Stimulation with phenylephrine (10 mumol/l) in Ca2(2+)-free medium caused a significantly reduced transient contraction in SHR as compared with control tissues. After pretreatment with CPA (30 mumol/l), this contraction was suppressed in SHR and WKY tissues. On the restoration of Ca2+, CPA induced a nifedipine (5 mumol/l) sensitive contraction, significantly larger in SHR than in WKY tissues. The relaxation rate to nifedipine of K(+)-induced contraction in CPA-treated SHR tissue was also reduced. We conclude that the ER Ca2+ pump in SHR aorta is less effective as compared with WKY tissue. The significantly greater CPA-induced contraction together with the reduced relaxation rate in the presence of CPA in SHR tissues as compared with controls suggests that Ca2+ handling was also altered at the plasma membrane.

Influence of internal calcium stores on calcium-activated membrane currents in smooth muscle.

Agonist-induced activation of second messenger systems that increase inositol 1,4,5-trisphosphate concentration plays an important role in the mobilisation of stored Ca2+ in smooth muscle. Release of Ca2+ which occurs spontaneously or by agonist stimulation causes activation of Ca(2+)-sensitive currents. Experiments using selective modulators of the Ca2+ uptake and release mechanisms of the sarcoplasmic reticulum provide evidence that depletion of the internal Ca2+ store triggers an influx of Ca2+ from the extracellular space. Recent data suggest that pool depletion causes the opening of non-selective cation channels which are possibly similar to those previously designated 'receptor-operated Ca2+ channels'. Ca2+ entry through these channels not only refills the internal Ca2+ pool, but also modulates membrane excitability by stimulating Ca(2+)-activated currents.

Morphological and functional characterization of vascular muscle cells enzymatically dispersed from dog mesenteric arteries.

The objective of this study was to compare the properties of single smooth muscle cells enzymatically dispersed from the dog mesenteric arteries to the properties of similar cells functioning in tissue strips. The isolated cells remained relaxed in nominally Ca(2+)-free medium for about 1-2 h after exposure to 1 mM Ca2+ and like intact mesenteric artery rings did not contract spontaneously. Enzymatically dispersed cells maintained all the characteristic morphological features observed in strips of muscle prior to isolation except that the amorphous materials covering the smooth muscle cell surfaces (basal lamina) were absent after enzymatic dispersion. Addition of 100 mM KCl to these vascular muscle cells elicited maximal shortening in the presence but not in the absence of extracellular Ca2+ and KCl-induced cell shortening was prevented by 10(-7) M nifedipine indicating the presence of functional voltage-operated Ca2+ channels. However, in contrast to the vascular muscle strips, in which graded contractile responses were observed with increasing KCl concentrations, isolated vascular muscle cells underwent nearly maximal contraction at concentrations as low as 15 mM KCl. Both intact tissue and isolated cell preparations responded similarly to phenylephrine in a concentration-dependent manner and the responses were blocked by prazosin. In contrast to muscle strips, the isolated cells did not shorten in response to phenylephrine in Ca(2+)-free medium. Isolated muscle shortened in the presence of sarcoplasmic reticulum selective Ca2+ transport ATPase inhibitors, cyclopiazonic acid or thapsigargin. Ryanodine also caused contraction. We conclude that enzymatically dispersed smooth muscle cells from dog mesenteric arteries are potentially useful for studies of the regulation of smooth muscle contractility, but have significantly increased sensitivity to external K+, implying an altered membrane potential or voltage dependence of ion channels. Their impaired ability to contract to phenylephrine in Ca(2+)-free medium implies some alteration in intracellular Ca2+ stores of their coupling to cellular activation. These differences will affect how the data obtained from freshly isolated enzymatically dispersed vascular muscle cells may be extrapolated to cell studies in intact tissues.

Characterization of alpha-adrenoceptors in canine mesenteric vein.

The alpha-adrenergic receptors (alpha-ARs) in canine mesenteric vein (DMV) were studied by using nonselective agonists and selective antagonists in functional studies and in ligand binding to classify the subtypes present. Based on functional studies of phenylephrine (PE)-induced contractions and ligand-binding interactions of [3H]-prazosin with prazosin (PR), WB 4101 (WB), 5-methylurapidil (5-MU), BMY 7378, and SK&F 105854, and pretreatment with chloroethylclonidine (CEC), DMV alpha1-ARs resembled the alpha1D subtype. However, the affinity of PR assessed in functional and ligand-binding studies was less (pK(B,D,i) < or = 9) than expected from previous characterization of cloned rat or human alpha1-AR (pKi > or = 10). Interactions with 5-MU, BMY 7378, or SK&F 105854 suggested the presence of some alpha1-ARs that were not typical of alpha1D-AR and that binding and functional interactions did not yield corresponding results. PR binding was abolished by treatment with CEC, contractile responses to PE were reduced in Emax, and the concentration-effect curve shifted to the left, as previously reported. DMVs contracted in response to alpha2-AR agonists and were studied when contractions were potentiated by increasing extracellular KCl to 20 mM. Rauwolscine (RAU) had K(B) values at these sites consistent with K(D) values in binding studies. CEC had no effect on RAU binding in DMV. Ligand-binding studies to [3H]-RAU sites did not reveal a clear identification of subtype, but these alpha2-ARs were clearly not alpha2B-ARs. We conclude that canine mesenteric vein contains alpha1D-like ARs, but with significant differences, and an unclassifiable alpha2-AR. There may also be a smaller population of other, not alpha1D-like ARs, receptors, mediating responses to PE and binding of prazosin.

Calretinin-immunoreactive neurons and their projections in the guinea-pig colon.

The distribution of nerve cells and fibres with immunoreactivity for the calcium-binding protein, calretinin, was studied in the distal colon of the guinea-pig. The projections of the neurons were determined by examining the consequences of lesioning the myenteric plexus. Calretinin-immunoreactive neurons comprised 17% of myenteric nerve cells and 6% of submucous nerve cells. Numerous calretinin-immunoreactive nerve fibres were located in the longitudinal and circular muscle, and within the ganglia of the myenteric and submucous plexuses. Occasional fibres were found in the muscularis mucosae, but they were very rare in the lamina propria of the mucosa. Lesion studies revealed that myenteric neurons innervated the underlying circular muscle and provided both ascending and descending processes that gave rise to varicose branches in myenteric ganglia. Calretinin-immunoreactive fibres also projected to the tertiary component of the myenteric plexus, and are therefore likely to be motor neurons to the longitudinal muscle. Varicose fibres that supplied the submucous ganglia appear to arise from submucous nerve cells. Arterioles of the submucous plexus were sparsely innervated by calretinin-immunoreactive fibres. The submucous plexus was the principal source of immunoreactive nerve fibres in the muscularis mucosae. This work shows that calretinin-IR reveals different neuronal populations in the large intestine to those previously reported in the small intestine.

Relative contributions of extracellular Ca2+ and Ca2+ stores to smooth muscle contraction in arteries and arterioles of rat, guinea-pig, dog and rabbit.

1. These studies describe the functional effects of modulation of the sarcoplasmic reticulum (SR) Ca2+ stores at three levels of the vasculature: (i) large arteries (rat and guinea-pig aorta); (ii) small resistance arteries (rat tail artery, rabbit mesenteric artery, dog mesenteric artery); and (iii) arterioles (guinea-pig submucosal arterioles of the small intestine). 2. All tissues responded to phenylephrine (PE; 10 mumol/L) with a transient contraction in Ca(2+)-free Krebs', reflecting Ca2+ release from PE-sensitive Ca2+ stores. After pretreatment with cyclopiazonic acid (CPA; 30 mumol/L) or thapsigargin (TSG; 1 mumol/L), putative SR Ca2+ pump inhibitors, the PE-induced contraction in a Ca(2+)-free medium was significantly inhibited in arterial tissues at all levels of the vasculature. Similarly, ryanodine (RYA; 30 mumol/L), an agonist that enhances Ca2+ release from the SR, also reduced the PE contraction in a Ca(2+)-free solution. 3. CPA or TSG alone in the presence of extracellular Ca2+, caused marked and sustained contraction in the rat and guinea-pig aorta and marked but transient or no contraction in the resistance arteries. In the rat and guinea-pig aorta, RYA caused a slowly developing tension. Little increase in basal tension was produced by RYA in resistance arteries and arterioles. 4. The findings show that an agonist-releasable Ca2+ pool is present at all levels of the vasculature that is independent of the size of the vessels and suggest that under normal physiological conditions there is an intimate balance between the roles of the plasma membrane and of the SR in the maintenance of vascular contractility. It appears that the role of the SR diminishes as the arteries become smaller, while Ca2+ fluxes across the plasma membrane predominates.

Thapsigargin inhibits repletion of phenylephrine-sensitive intracellular Ca++ pool in vascular smooth muscles.

Thapsigargin (TSG), a putative selective Ca(++)-ATPase inhibitor, has been used to study Ca++ mobilization in many non-excitable cell types. This study aims to determine whether TSG is effective as a selective microsomal Ca++ uptake inhibitor by studying its ability to affect repletion of the phenylephrine (PE)-sensitive Ca++ pool in rat aorta and dog mesenteric artery evaluated by contractility studies. TSG caused a concentration-dependent contraction that was dependent on the concentration of extracellular Ca++. Ca++ influx promoted by TSG was found to occur mostly through L-type Ca++ channels in the dog mesenteric artery but not in the rat aorta. When arterial rings, depleted of their PE-sensitive internal store, were allowed to replete their stores in normal Krebs' solution or in the presence of elevated K+ levels, it was found that repletion was significantly enhanced in the presence of elevated K+. In TSG-treated rings, however, repletion was significantly inhibited under both conditions as indicated by the subsequent PE-induced contraction in Ca(++)-free medium. While the rate of contraction induced by elevated K+ levels was slow immediately after pool depletion in controls, it was rapid in TSG-treated arterial rings. The slow onset of K+ contraction may reflect Ca++ uptake into the pool which was absent in TSG-treated arteries. Differences in the behavior of the two arteries were noted and these may reflect differences in the size of their Ca++ store and their coupling to the extracellular space. Single cells isolated from the dog mesenteric artery were also found to shorten in response to TSG to an amount comparable with that obtained from whole tissue experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Ryanodine and the adrenergic, purinergic stimulation in the rat vas deferens smooth muscle: functional and radioligand binding studies.

The distribution of [3H]ryanodine binding in subcellular fractions isolated from rat vas deferens (RVD) paralleled that of NADPH cytochrome C reductase activity indicating an endoplasmic reticulum origin of the binding sites. Scatchard analysis of [3H] ryanodine binding indicated an homogenous site with a Kd value of 6.4 nM. The maximum number of ryanodine binding sites was 488 fmol of [3H]ryanodine per milligram of protein. Norepinephrine (NE) or ATP endogenously released after electrical field stimulation (tetrodoxin-sensitive responses), both produced a biphasic contraction of the RVD when the action of the other was blocked. When NE was the agonist (prazosin-sensitive response), the initial transient contraction was suppressed by 30 microM ryanodine whereas the secondary component was abolished by 2 microM nifedipine. When ATP was the agonist (P2 tachyphylaxis-sensitive response), both phases of the contraction were suppressed by 2 microM nifedipine. However, the initial phasic component of the contraction induced by endogenously released ATP was also inhibited by 30 microM ryanodine except at high stimulation frequency (10 Hz). Exogenously added NE and alpha, beta methylene ATP produced concentration-dependent contractions of the RVD. The effect of both agonists was inhibited by 2 microM nifedipine whereas 30 microM ryanodine had little effect except at high concentrations of NE. We conclude that ryanodine binding sites reside in RVD endoplasmic reticulum. There was a lack of uniformity in the effect of ryanodine and nifedipine against alpha adrenoceptor stimulation and purinoceptor stimulation indicating a difference in the stimulation-contraction coupling process between these two modes of stimulation.

Pharmacological and immunocytochemical characterization of subtypes of alpha-1 adrenoceptors in dog aorta.

In this study, the effects of nine alpha-1 adrenoceptor antagonists [prazosin, WB 4101 (WB), chloroethylclonidine (CEC), 5-methylurapidil (5-MU), BMY 7378 (BMY), MDL 73005EF (MDL73), MDL 72832 (MDL72), RS 17053 (RS) and SK&F 105854 (SKF)] were studied on contractile responses to phenylephrine (PE) of the endothelium-denuded dog aorta in vitro. All antagonists, except CEC, 5-MU and RS, produced concentration-dependent competitive inhibition of contractile responses of the aorta to PE. The rightward shift of the concentration-response curves of PE yielded constant pKB values with increasing antagonist concentrations in most cases allowing a single pooled value to be determined: for prazosin, a pKB of 8.99 +/- 0.11 (n = 20, KB of 1.03 nM); for WB, a pKB of 8.75 +/- 0.08 (n = 23, KB of 1.76 nM); for BMY, a pKB of 7.21 +/- 0.13 (n = 13, KB of 62 nM); for MDL72, a pKB of 7.95 +/- 0.15 (n = 12, KB of 11.2 nM); and for SK&F 105854, a pKB of 5.82 +/- 0.08 (n = 15, KB of 1.52 microM). For MDL73, pKB values decreased with antagonist concentration: 7.88 +/- 0.06 at 10 nM, 7.56 +/- 0.28 at 100 nM and 6.92 +/- 0.18 at 1000 nM, which suggests the presence of more than one receptor subtype. CEC (10 and 100 microM) almost completely inhibited responses to PE; lower concentrations had no significant effect. 5-MU (10-300 nM) and RS (3-300 nM) were ineffective antagonists in this tissue. Because WB, a highly selective alpha-1D and alpha-1A adrenoceptor subtypes inhibitor, blocked PE responses (with less affinity than for alpha-1A adrenoceptors), and 5-MU and RS, which are selective blockers for alpha-1A adrenoceptor, were ineffective, we conclude that alpha-1A adrenoceptors are absent in the dog aorta. The effects of the less selective MDL72 were inconsistent with actions at alpha-1B or alpha-1D adrenoceptors. Although WB shifted the PE concentration-response curve to the right, the abilities of BMY, MDL73 and SKF to inhibit competitively PE contraction were of lower affinity compared with expectations for interaction with alpha-1D adrenoceptors; they are not the predominant subtype. The complete inhibition of PE responses by CEC suggests that the dog aorta contains the alpha-1B adrenoceptor subtype. In immunocytochemical studies of the expression of alpha-1B adrenoceptor, all cells apparently expressed this protein. Moreover, Western blot studies of the microsomal fractions confirmed the presence of alpha-1B adrenoceptors. In the dog aorta, the alpha-1 adrenoceptors predominantly resemble alpha-1B rather than alpha-1D adrenoceptors as reported in the rat aorta.