Relationship between 45Ca movements, different calcium components and responses to acetylcholine and potassium in tracheal smooth muscle

Correlations between tension responses elicited with acetylcholine (ACh) and high K+ and corresponding alterations in Ca++ mobilization were obtained in rabbit and canine tracheal smooth muscle. Removal of Ca++ or preincubation with D-600 (50 microM) inhibited responses to K+ (50 or 80 mM) and low ACh (89 nM) and had only a small effect on responses to high ACh (8.9 microM). Conversely, solutions containing Sr++ instead of Ca++ inhibited responses to both concentrations of ACh to a greater degree than were those to K+. Washout of slow component 45Ca into a O-Ca solution was more rapid in rabbit trachea than reported previously for rabbit aorta. Washout of tracheal smooth muscle into an 80.8 mM La -substituted solution at 0.5 degrees C removed superficial (La -accessible) 45Ca and blocked both 45Ca uptake and most 45Ca efflux. D-600, which had no significant effect on control 45Ca uptake in rabbit aortic smooth muscle, decreased 45Ca uptake by 33% in rabbit tracheal smooth muscle. The uptake of 45Ca from the Ca++ binding sites with low affinity for Ca++ was increased by 80 mM K+, 50 mM K+ or 8.9 microM ACh, and the accumulation of Ca++ from the Ca++ binding sites with high affinity for 45Ca was inhibited by Sr++. The stronger effect of either Ca++ removal or D-600 on responses to K+ and the correspondingly greater effect of Sr++ on responses to ACh indicate that different Ca++ stores are present in tracheal smooth muscle. These Ca++ components appear to be qualitatively similar to those present in aortic smooth muscle but they differ quantitatively and are not as readily dissociated as are aortic Ca++ components.     

Inhibitory effects of a synthetic atrial peptide on contractions and 45Ca fluxes in vascular smooth muscle

The mechanism of a synthetic atrial peptide (APII)-induced inhibition of smooth muscle contractility was investigated by studying its effects on tension development and 45Ca fluxes in isolated rabbit aorta. APII (10(-9) to 10(-7) M) produced a dose-dependent relaxation of contractions produced by alpha adrenoceptor activation with norepinephrine (NE; 10(-6) M). APII was a potent relaxant of NE contraction with an IC50 = 1.1 X 10(-8) M, with 10(-7) M APII causing a 97% relaxation. APII also produced a dose-dependent inhibition of NE contraction when added to the resting muscle before the exposure to NE. The relaxing effects of APII were found to be endothelium independent. In contrast, APII was only marginally effective in relaxing high-K+ contraction, with 10(-7) M APII causing only 17% relaxation. Furthermore, when a NE contraction was obtained on top of a high-K+ contraction, APII was still capable of relaxing the NE component. APII was similarly more effective in inhibiting NE-stimulated 45Ca influx than high-K+-stimulated 45Ca influx, indicating selective action of APII on the receptor-operated Ca++ channels. This was in contrast to D600, a well known Ca++ antagonist, which had a more selective inhibitory effect on the potential-operated Ca++ channels. The data presented indicate that APII is a potent relaxant of contractions produced by receptor-agonists involving 45Ca influx through receptor-operated Ca++ channels. APII may also prove to be a very useful tool to further distinguish and define receptor-operated Ca++ channels and potential-operated Ca++ channels in vascular smooth muscle.     

Insulin attenuates agonist-mediated calcium mobilization in cultured rat vascular smooth muscle cells.

Insulin has been shown to attenuate pressor-induced vascular contraction, but the mechanism for this vasodilatory action is unknown. This study examines the effect of insulin on angiotensin II (ANG II)-induced increments in cytosolic calcium in cultured rat vascular smooth muscle cells (VSMC). 20-min incubations with insulin (10 microU/ml to 100 mU/ml) did not alter basal intracellular calcium concentration ([Ca2+]i), but inhibited the response to 100 nM ANG II in a dose-dependent manner (ANG II alone, 721 +/- 54 vs. ANG II + 100 mU/ml insulin, 315 +/- 35 nM, P < 0.01). A similar effect of insulin on ANG II action was observed in calcium poor buffer. Moreover, insulin did not effect calcium influx. ANG II receptor density and affinity were not affected by 24-h incubation with insulin. To further clarify the mechanisms of these observations, we measured ANG II-induced production of inositol 1,4,5-triphosphate (IP3), and IP3-releasable 45Ca. Insulin treatment did not alter ANG II-stimulated IP3 production. However, IP3-stimulated release of 45Ca in digitonin permeabilized cells was significantly reduced after 5-min incubations with 100 mU/ml insulin. Thapsigargin induced release of calcium stores was also blocked by insulin. Thus, insulin attenuates ANG II-stimulated [Ca2+]i primarily by altering IP3-releasable calcium stores. Insulin effects on ANG II-induced [Ca2+]i were mimicked by preincubation of VSMC with either sodium nitroprusside or 8-bromo-cGMP. As elevations in cGMP in vascular tissue lower [Ca2+]i, it is possible that insulin affects IP3 release of calcium by a cGMP-dependent mechanism that would contribute to its vasodilatory effects.     

Serotonin-induced Na+/K+ pump stimulation in vascular smooth muscle cells. Evidence for coupling to multiple receptor mechanisms.

Exposure of cultured canine femoral artery vascular smooth muscle cells to serotonin (5-HT) caused a 3.6-fold stimulation of ouabain-sensitive 86Rb uptake. The 5-HT2 receptor antagonist, ketanserin, partly blocked the 5-HT-mediated Na+/K+ pump stimulation and the 5-HT1/5-HT2 receptor antagonist, methiothepin, completely blocked the response, suggesting that both 5-HT1 and 5-HT2 receptors play a role in the 5-HT-mediated Na+/K+ pump activation. Second messengers generated by 5-HT2 receptor-mediated phosphoinositide hydrolysis, Ins(1,4,5)P3 and diacylglycerol were implicated in the stimulatory action of 5-HT on the vascular Na+/K+ pump. Like some other contractile agonists, 5-HT activated a Na+ influx pathway which caused Na+/K+ pump stimulation by increasing the rate-limiting substrate. The maximum stimulation of Na+ influx by 5-HT was 2.5-fold. The 5-HT-stimulated Na+ influx was totally blocked by methiothepin but only 29% inhibited by ketanserin, indicating that most of the Na+ influx was mediated by the 5-HT1 receptor. The 5-HT-stimulated Na+ influx was substantially inhibited by 50 microM dimethylamiloride, suggesting that the Na+ influx pathway stimulated by 5-HT was Na+/H+ exchange. BAPTA/AM 1,2-[bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra (acetoxymethyl) ester], an intracellular Ca++ chelator, partly blocked 5-HT-stimulated Na+ influx and ouabain-sensitive 86Rb uptake, suggesting that Ca++ is an important mediator of these responses. These data suggest that: 1) 5-HT, in addition to its well known activity as a contractile agonist, can stimulate the electrogenic Na+/K+ pump which, in theory, would tend to oppose contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of amiloride-sensitive apical Na+ conductance by acetylcholine in rabbit cortical collecting duct perfused in vitro.

We examined effects of acetylcholine (ACh) on the electrical parameters and intracellular Ca2+ concentration ([Ca2+]i) in the isolated rabbit cortical collecting duct (CCD) perfused in vitro using the conventional microelectrode technique and microscopic fluorescence spectrophotometry. ACh (10(-8) to 10(-5) M) in the bath caused a positive deflection of the transepithelial voltage (VT) and an increase in [Ca2+]i. Carbachol also showed similar but smaller effects. The effects of ACh were antagonized by muscarinic receptor antagonists. ACh at 10(-6) M hyperpolarized the apical membrane voltage and increased the fractional resistance of the apical membrane of the collecting duct cells accompanied by a positive deflection of VT and an increase in transepithelial resistance, whereas it did not affect these parameters in the beta-intercalated cells. In the presence of 10(-5) M amiloride in the lumen, the effects of ACh were almost completely abolished. The ACh-induced increase in [Ca2+]i is accounted for by the release of Ca2+ from intracellular store and Ca2+ entry from the bath. In the absence of Ca2+ in the bath, the ACh-induced changes in electrophysiological parameters were significantly smaller than those observed in the presence of Ca2+. Both phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutylate (PDBu), activators of protein kinase C (PKC), also inhibited the apical Na+ conductance. In the presence of PMA or PDBu in the bath, ACh did not show further inhibitory effect. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine, an inhibitor of PKC, partially attenuated the effect of ACh. These observations indicate that ACh inhibits the apical Na+ conductance partly by both increasing [Ca2+]i and activating PKC. Such an action of ACh may partially explain its natriuretic effect.     

Effects of tacrine on insulin secretion and 86Rb+ and 45Ca++ efflux from rat pancreatic islets.

Tacrine (1,2,3,4-tetrahydro-9-aminoacridine), a drug that has attained interest because of its ability to alleviate symptoms in Alzheimer's type of dementia, was found to stimulate insulin secretion from isolated rat pancreatic islets. The insulinotropic effect of the drug was observed at 8.3 mM but not at 3.3 mM glucose and was dependent on extracellular Ca++. From perifused 86Rb(+)-prelabeled islets, tacrine inhibited the fractional efflux of 86Rb+ at 3.3 mM glucose, but stimulated 86Rb+ efflux at 8.3 mM glucose. These effects persisted in the absence of extracellular Ca++. Tacrine also stimulated 45Ca++ efflux from perifused 45Ca(++)-prelabeled islets at 8.3 mM but had no effect on 45Ca++ efflux at 3.3 mM glucose or in the absence of extracellular Ca++. It is concluded that tacrine potentiates glucose-stimulated insulin secretion by a mechanism that is dependent on extracellular Ca++ and involves an increased Ca++ influx. The increased Ca++ influx is either secondary to a decreased K+ permeability induced by an inhibition of ATP-dependent K+ channels and/or due to a direct effect of tacrine on glucose-activated Ca++ channels.     

Ca++ utilization in the constriction of rat aorta to stimulation of protein kinase C by phorbol dibutyrate

To determine the role of activated protein kinase C in vascular smooth muscle contraction, phorbol dibutyrate was used to stimulate this enzyme in order to evaluate the source(s) of Ca++ (10(-8) to 3 X 10(-6) M) elicited a concentration-dependent sustained contraction which was slow in onset but progressive in developed tension. The maximal contractile response induced by phorbol dibutyrate was only partly dependent on influx of extracellular Ca++ as shown by similar reductions (40%) produced by Ca++-free buffer, LaCl3 (1 mM) or nifedipine (10(-6) M). These data suggest that phorbol dibutyrate is able to open Ca++ channels which are sensitive to nifedipine blockade. However, unlike norepinephrine or K+-depolarization, phorbol dibutyrate evoked a slow 45Ca++ influx which occurred only after extended contact time. Pretreatment with nifedipine again abolished this response. In contrast to norepinephrine, phorbol dibutyrate did not cause 45Ca++ efflux indicating that intracellular Ca++ was not mobilized. It is concluded that the residual 60% contraction to phorbol dibutyrate most likely occurs via a mechanism independent of the Ca-calmodulin pathway.     

Reversible inhibition of acetylcholine contracture of molluscan smooth muscle by heavy metals: correlation to Ca++ and metal content

The present study examined the effects of three heavy metals on the acetylcholine (ACh) contracture and Ca++ kinetics of the anterior byssus retractor muscle of Mytilus edulis. An isolated tissue bioassay using anterior byssus retractor muscle was prepared according to standard procedures and the isometric tension produced in response to ACh was measured. Ten millimolar Ni++, Co++ or Cd++ reduced the maximum contracture response to ACh in zero-Ca medium in a time-dependent manner. The inhibition was reversed upon restoration of medium containing 10 mM Ca++. The loss (and re-establishment) of contracture response to ACh corresponded to the influx (and efflux) of the heavy metal ions opposite to the direction of Ca++ flow. These results are consistent with the concept that the loss of the ACh contracture response is attributable to the displacement of tissue Ca++ from release sites by heavy metals.     

Calcium ion requirement for acetylcholine-stimulated breakdown of triphosphoinositide in rabbit iris smooth muscle.

Previous studies from this laboratory have established that addition of acetylcholine (ACh) or norepinephrine to 32P-labeled rabbit iris smooth muscle increases significantly the breakdown of triphosphoinositide (TPI) and that these stimulatory effects are blocked by atropine and phentolamine, respectively. The present studies were undertaken in order to show the effect of Ca++ on the ACh-stimulated breakdown of TPI ("TPI effect") in this tissue. Paired iris smooth muscles were prelabeled with 32Pi for 30 minutes at 37 degrees C in Ca++-free iso-osmotic salt medium. The prelabeled irises were then washed and incubated for 10 minutes in nonradioactive Ca++-free medium which contained 10 mM 2-deoxyglucose under various conditions. The phospholipids were isolated by means of two-dimensional thin-layer chromatography and their radioactivities were determined. In the absence of Ca++, 50 micrometer ACh increased TPI breakdown and phosphatidic acid (PA) labeling by 16 and 38%, respectively. In the absence of ACh, 0.75 micrometer Ca++ increased TPI breakdown and PA labeling by 11 and 20%, respectively. When both ACh and Ca++ were added, the increase in TPI breakdown and PA labeling rose to 32 and 74%, respectively. The labeling of phosphatidylinositol was found to be insensitive to the presence of Ca++. Ca++ was determined in the iris smooth muscle and it was found to contain 3.13 mumol of Ca++ per g of tissue. This was reduced by 80% after the muscle was washed and incubated in a medium which contained 0.25 micrometer ethyleneglycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA). The TPI effect was abolished by 0.25 micrometer EGTA and restored when excess Ca++ (1.25 micrometer) was added. Concentrations of Ca++ as low as 50 micrometer provoked a TPI effect. Sr++ (2 micrometer), but not Ba++ or Mn++, was found to substitute partially for Ca++. Ionophore A-23187 (20 micrometer) was found to increase the breakdown of TPI and labeling of PA by 11 and 24%, respectively. High concentrations of Ca++ (20 mM) exerted similar effects. The increase in TPI breakdown and PA labeling in response to these agents, in contrast to the TPI effect in response to ACh, was not blocked by atropine. This suggests that the observed effects are not caused by the release of endogenous ACh from the muscle. A possible interpretation for the above observations on the role of Ca++ in the TPI effect at the postsynaptic membrane of the iris smooth muscle could be: (formula: see text).     

Inhibition of ouabain-induced increase in Na content of cultured myocardial cells by quinidine and procainamide

Addition of ouabain caused gradual increases of both the Na content of cultured myocardial cells and the rate of Ca++ uptake by the cells. Ouabain-induced irregular beating of the cells (ouabain toxicity) appeared to develop when the Na content and the rate of Ca++ uptake exceeded about 1.5 and 2.0 times, respectively, the normal levels. Quinidine and procainamide prevented ouabain-induced increases of the Na content and the rate of Ca++ uptake as well as ouabain-induced toxicity. The problem of how quinidine and procainamide counteract the effects of ouabain was then studied. Quinidine and procainamide did not affect the Na+-Ca++ exchange activity. Na+,K+-adenosine triphosphatase activity, Na+-pumping out activity or ouabain-binding activity of myocardial cells, but inhibited passive Na+ influx, which is achieved by a simple diffusion system. From these observations, it is suggested that inhibition by quinidine or procainamide of passive Na+ influx indirectly prevents ouabain-induced increase in the intracellular Na content of myocardial cells and that this presumably explains at least in part the inhibitory effect of quinidine and procainamide on ouabain-induced irregular beating.     

Noncontractile acetylcholine receptor-operated Ca++ mobilization: suppression of activation by open channel blockers and acceleration of desensitization by closed channel blockers in mouse diaphragm muscle

The effects of various blockers of the nicotinic acetylcholine receptor-activated ionic channel on noncontractile slow Ca++ mobilization were investigated at the neuromuscular synapse of aequorin-injected diaphragm muscles of mice. Intracellular Ca++ mobilization (Ca++ transients) was evoked in the presence of neostigmine (0.3 microM) by nerve stimulation. Bupivacaine, an open channel blocker, decreased the peak amplitude, whereas chlorpromazine, a closed channel blocker, shortened the duration. Phencyclidine, an open and closed channel blocker, decreased both peak amplitude and duration. beta-Eudesmol, a compound of Atractylodes lancea, clearly and specifically shortened the duration but had little effect on peak amplitude. All the above channel blockers, when given in the same concentration ranges, also blocked the total amount of contractile Ca++ transients. The effects of bupivacaine, chlorpromazine and phencyclidine on noncontractile Ca++ transients were not affected by 5 mM [Ca++]o, whereas the effect of beta-eudesmol was enhanced. Geographutoxin II (0.3 microM), a skeletal muscle Na+ channel blocker, selectively and partly reversibly blocked contractile Ca++ transients without affecting noncontractile ones. These results suggest that: 1) the activation of noncontractile Ca++ mobilization is suppressed by open channel blockers, whereas its desensitization is accelerated by closed channel blockers and 2) activation of the muscle Na+ channel and subsequent release of Ca++ from sarcoplasmic reticulum is not involved in the mechanism of noncontractile Ca++ mobilization. It may reflect the steps of the desensitization process.     

Characterization of K+ channel-dependent as well as -independent components of pinacidil-induced vasodilation

The mechanisms of pinacidil-induced direct vasodilation were studied in vitro in RMA and RAO. In RMA, pinacidil produced dose-dependent relaxations of norepinephrine (5 microM)-induced contractions with an IC50 of 0.2 microM. This component of pinacidil relaxation appeared to be dependent on K+ conductance because pretreatment with tetraethylammonium (10 mM), Ba++ (0.5 mM), glyburide (1 microM) and 20 mM K+ all caused a rightward shift of the pinacidil dose-response curve (DRC) and a corresponding increase in the pinacidil IC50. However, additional relaxation effects of pinacidil were still evident in the presence of various K+ channel blockers. Pinacidil also showed a relaxation DRC under the condition of 80 mM K+ contraction in both RMA and RAO with IC50 values of 27 and 50 microM, respectively. Pinacidil could also produce maximal relaxation in RMA and RAO remained unaffected in 145 mM K+ (zero Na+) depolarizing solution suggesting a lack of dependence on Na(+)-Ca++ exchange mechanism for this action of pinacidil. Studies using 1 or 3 min pulse labeling with 45Ca showed an absence of an inhibitory effect of pinacidil (at 50 and 100 microM) on unidirectional 45Ca influx stimulated by high-K+. Net 45Ca uptake studies showed that pinacidil inhibited high-K+ stimulated 45Ca uptake at 100 but not at 50 microM. Ryanodine (10-100 microM) was used as a tool to investigate the role of sarcoplasmic reticulum (SR) in this action of pinacidil. Under the condition in which ryanodine (10-100 microM) treatment was found to cause the SR to be nonfunctional, pinacidil relaxation DRC remained unaltered, suggesting a lack of a stimulatory effect of pinacidil on SR Ca++ accumulation. These data thus show that the K+ channel-independent effect of pinacidil does not involve to any significant degree an effect of pinacidil on plasmalemmal voltage-sensitive Ca++ channels, SR Ca++ stores, Na(+)-Ca++ exchange or membrane hyperpolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium channel blockers: correlation among receptor binding, calcium uptake and contractility in vitro

Ten known calcium channel blockers were studied for inhibition of K+-induced 45Ca++ uptake into rabbit aortic smooth muscle cells in culture, and for displacement of [3H]nitrendipine [2,6-dimethyl-3-carbomethoxy-5-carbomethoxy-4-(3-nitro)phenyl-1, 4-dihydroxypyridine] binding to rat ventricular membrane preparations, in order to relate their effects on receptor binding with their inhibitory activities on 45Ca++ uptake and on contractile responses of vascular smooth muscle. Steady-state 45Ca++ uptake increased with K+ concentration in a dose-dependent manner. With 25 to 50 mM K+, Ca++ uptake was 0.6 nmol of Ca++ per one million cells. All calcium channel blockers inhibited K+-induced 45Ca++ uptake and [3H]nitrendipine binding in a dose-dependent fashion. The enatiomeric dihydropyridines 202-791 [isopropyl-4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2, 6-dimethyl-5-nitro-3-pyridinecarboxylate] exhibited marked stereoselectivity in both studies, the agonist (+)-202-791 significantly enhancing 45Ca++ uptake at 15 to 50 mM K+. The similarity between the order of potency in inhibiting 45Ca++ uptake and displacing [3H]nitrendipine resulted in a highly significant linear (1:1) correlation. An equally significant correlation was also established for the 10 blockers between their inhibitory potencies on 45Ca++ uptake and the contractile response of rabbit aortic strips as cited in the literature. These findings support the hypothesis that calcium channel blockers block contraction of vascular muscle by inhibiting cellular calcium uptake through voltage-dependent calcium channels as a result of binding to receptors associated with these channels. The aortic cells possess channels that are functionally similar to those found in intact vascular tissue.     

Receptor-coupled G proteins mediate contraction and Ca++ mobilization in isolated intestinal muscle cells.

Guanosine 5'-O-(gamma-thio)triphosphate (GTP[S]), NaF and cholecystokinin-octapeptide (CCK-8) were used to examine the participation of G proteins in agonist-induced contraction of smooth muscle cells isolated separately from circular and longitudinal muscle layer of guinea pig intestine. All three agents stimulated inositol 1,4,5-triphosphate (InsP3) production and protein kinase C activity to the same extent in permeabilized (GTP[S] and CCK-8) and nonpermeabilized (NaF and CCK-8) muscle cells. InsP3 production was 9 to 13 times higher in circular muscle cells consistent with preferential hydrolysis of phosphatidylinositol 4,5-biphosphate in this cell type. InsP3 production and protein kinase C activation in permeabilized muscle cells were abolished by guanosine 5'-O-(beta-thio)diphosphate (10 microM). Maximal concentrations of GTP[S] (100 microM), CCK-8 (1 nM) and InsP3 (1 microM) elicited similar increases in [Ca++]i, net 45Ca++ efflux and contraction in permeabilized circular, but not longitudinal, muscle cells [( Ca++]i: 224 +/- 35 nM, 279 +/- 29 nM and 288 +/- 45 nM increase above basal level; 45Ca++ efflux: 35 +/- 2%, 34 +/- 3% and 37 +/- 3% decrease in cell Ca++ content; contraction: 26 +/- 2%, 24 +/- 2% and 25 +/- 2% decrease in cell length). The responses to GTP[S] and CCK-8 were abolished by guanosine 5'-O-(beta-thio)diphosphate (10 microM) and heparin (10 micrograms/ml), whereas the response to InsP3 was abolished by heparin only. Maximal concentrations of NaF and CCK-8 elicited similar increases in [Ca++]i and contraction in nonpermeabilized circular and longitudinal muscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Feedback inhibition of cyclic adenosine monophosphate-stimulated Na+ transport in the rabbit cortical collecting duct via Na(+)-dependent basolateral Ca++ entry.

Arginine vasopressin (AVP) transiently stimulates Na+ transport in the rabbit cortical collecting duct (CCD). However, the sustained effect of both AVP and its putative second messenger, cyclic adenosine monophosphate (cAMP), on Na+ transport in the rabbit CCD is inhibitory. Because maneuvers that increase [Ca++]i inhibit Na+ transport, the effects of AVP and cell-permeable cAMP analogues, on [Ca++]i were investigated in fura-2-loaded in vitro microperfused rabbit CCDs. Low-dose AVP (23-230 pM) selectively stimulated Ca++ influx, whereas 23 nM AVP additionally released calcium from intracellular stores. 8-chlorophenylthio-cAMP (8CPTcAMP) and 8-bromo-cAMP (8-Br-cAMP) also increased CCD [Ca++]i. The 8CPTcAMP-stimulated [Ca++]i increase was totally dependent on basolateral [Ca++]. In the absence of cAMP, peritubular Na+ removal produced a marked increase in [Ca++]i, which was also dependent on bath [Ca++], suggesting the existence of basolateral Na+/Ca++ exchange. Luminal Na+ removal in the absence of cAMP did not alter CCD [Ca++]i, but it completely blocked the cAMP-stimulated [Ca++]i increase. Thus the cAMP-dependent Ca++ increase is totally dependent on both luminal Na+ and basolateral Ca++, suggesting the [Ca++]i increase is secondary to cAMP effects on luminal Na+ entry and its coupling to basolateral Na+/Ca++ exchange. 8CPTcAMP inhibits lumen-to-bath 22Na flux [JNa(l-b)] in CCDs bathed in a normal Ca++ bath (2.4 mM). However, when bath Ca++ was lowered to 100 nM, a maneuver that also blocks the 8CPTcAMP [Ca++]i increase, 8CPTcAMP stimulated, rather than inhibited JNa(l-b). These results suggest that cAMP formation initially stimulates CCD Na+ transport, and that increased apical Na+ entry secondarily activates basolateral Ca++ entry. The cAMP-dependent [Ca++]i increase leads to inhibition Na+ transport in the rabbit CCD.     

Block of 45Ca uptake into synaptosomes by methylmercury: Ca++- and Na+-dependence

Block of Ca++ influx into isolated nerve terminals by the neurotoxicant methylmercury (MeHg) was studied for its dependence on extracellular Ca++ and Na+. Depolarization-independent entry of 45Ca++ was determined in rat forebrain synaptosomes incubated in 5 mM K+ solution. 45Ca++ uptake was similarly measured after 1 ("fast" phase) or 10 sec ("total") of elevated K+ (41.25 mM)-induced depolarization or after 10 sec of elevated K+-induced depolarization after synaptosomes had been predepolarized for 10 sec in Ca++- and MeHg-free solutions ("slow" phase). In 5 mM K+ solutions, MeHg concentrations of 125 microM and greater significantly reduced synaptosomal 45Ca++ uptake measured during 1 or 10 sec of incubation. In K+-depolarized synaptosomes, the estimated IC50 for block of total, fast and slow 45Ca++ uptake by MeHg is 75 microM; 250 microM MeHg reduced uptake by approximately 90%. The reversibility of block by extracellular Ca++ was tested by increasing the extracellular Ca++ concentration from 0.01 to 1.15 mM. When compared to control, 50 microM MeHg reduced total uptake of 45Ca++ by greater than or equal to 70% and reduced fast uptake by 20 to 60% at all concentrations of extracellular Ca++ tested. At Ca++ concentrations of 0.01 to 0.15 mM, MeHg (50 microM) reduced slow uptake by 75 to 90%, but did not affect slow uptake at higher Ca++ concentrations (greater than or equal to 0.30 mM). When the dependence of block of 45Ca++ uptake on extracellular Na+ was tested, equivalent levels of inhibition were caused by MeHg (25 microM) for fast uptake by synaptosomes in Na+-containing and Na+-free solutions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological properties of potassium currents in swine tracheal smooth muscle.

The pharmacological properties of large conductance Ca(++)-activated K+ channels and spontaneous transient outward currents (STOCs) in swine tracheal smooth muscle (TSM) were studied using enzymatically dissociated single cells and patch clamp techniques. Recording from inside-out patches showed that extracellular tetraethylammonium caused a concentration-dependent decrease in single channel current amplitude. Acetylcholine (ACh; 10(-8) M) induced repetitive outward current transients in cell-attached patches due to simultaneous opening of many large conductance Ca(++)-activated K+ channels. Whole-cell recordings revealed the presence of STOCs in TSM membrane that were inhibited by tetraethylammonium at concentrations similar to those which inhibited the large conductance Ca(++)-activated K+ channels. Caffeine (5 mM) stimulated and then inhibited STOCs, suggesting that they are induced by the release of calcium from internal stores. ACh (1 microM) initially increased STOC frequency followed by an inhibition of STOCs. The effects of ACh occurred in control solution (2 mM Ca++) or when calcium influx was eliminated (0 Ca++, 2 mM Mn++). ACh also induced an inward current at negative membrane potentials associated with an increase in conductance. We conclude that inhibition of STOCs (i.e., potassium channel activity) and induction of an inward cation current by ACh may give rise to the depolarization observed in the presence of ACh and be important in the development of ACh-induced contractions in TSM.     

Electrophysiologic and biochemical studies on the putative Ca++ channel blocker MDL 12,330A in an endocrine cell

MDL 12,330A is a molecule structurally unrelated to other organic Ca++ channel ligands that may alter Ca++ channel function. Using whole cell patch clamp, [3H]PN200-110 binding and 45Ca++ uptake studies, we examined the effects of this compound on voltage-dependent Ca++ channels in rat anterior pituitary cells. At a concentration of 10(-5) M, MDL 12,330A showed little effect on outward K+ current, Na+ current or low-threshold Ca++ current in this cell line. At 10(-6) M, MDL 12,330A reversibly inhibited slow Ca++ current in a voltage-dependent manner. 45Ca++ uptake was also blocked by this compound at 10(-6) M, whereas [3H]PN200-110 binding was stimulated at concentrations of 10(-7) to 10(-6) M. The results are consistent with an interaction of MDL 12,330A with slow Ca++ channels at a site allosterically linked to the 1,4-dihydropyridine binding site.     

Inhibition of tracheal smooth muscle contraction and myosin phosphorylation by ryanodine

Previous studies have shown that muscarinic activation of airway smooth muscle in low Ca++ solutions increases myosin phosphorylation without increasing tension. Blocking Ca++ influx reduced phosphorylation, but not to basal levels. It was proposed that release of intracellular Ca++ contributed to dissociation of phosphorylation and contraction. To test this hypothesis the effects of ryanodine were studied under similar conditions. Ryanodine (10(-7) to 10(-5) M) antagonized caffeine-induced contraction of canine tracheal smooth muscle. Ryanodine also reduced carbachol-induced contractions and carbachol-induced myosin phosphorylation. The effect of ryanodine on potassium and serotonin-induced contractions was also investigated to test for a nonspecific inhibitory effect. In contrast to the effect on carbachol responses, ryanodine (10(-5) M) potentiated the contractile response to low concentrations of serotonin and potassium, but had no effect on the maximum response to either stimulant. Carbachol (10(-6) M) and ryanodine (10(-5) M) both significantly decreased 45Ca++ content of tracheal muscle. The effect of ryanodine and carbachol together on 45Ca++ content was not greater than either drug alone suggesting that ryanodine reduces the caffeine and carbachol responses by depleting releaseable Ca++ stores. Ryanodine significantly reduced Ca++-induced contraction and myosin phosphorylation in carbachol-stimulated muscle, suggesting that some of the Ca++ responsible for elevated phosphorylation is released from the sarcoplasmic reticulum.     

Effect of canrenone on the disturbances of cation handling induced by ouabain in macrophages and vascular smooth muscle cells

Ouabain induced rapid and profound modifications of Na+ and K+ contents in mouse macrophages and cultured vascular smooth muscle cells. In mouse macrophages, we found a one-to-one net Na+ gain and K+ depletion, with a maximal initial rate of 30 to 35 mmol (l X cells X hr)-1 and with an IC50 of about 100 microM. The one-to-one exchange results from at least two additive effects: inhibition of the Na+,K+-pump and stimulation of a furosemide-sensitive, outward Na+,K+-cotransport by the increase in internal Na+ content. The latter effect helps the cell to maintain a normal cell volume in spite of the large changes in internal cation content. In cultured vascular smooth muscle cells from rat aorta, ouabain provoked net Na+ gain and stimulated a quinidine-sensitive, K+-efflux. This likely reflects the opening of Ca+-dependent, K+-channels in response to an increase in cytosolic-free Ca+ content. Canrenone, an antihypertensive drug, has been shown to behave like a partial agonist at the digitalisreceptor site of the Na+,K+-pump. We observed here in mouse macrophages and cultured vascular smooth muscle cells that: canrenone alone (or at low ouabain concentrations) induces slight Na+ gain and K+ depletion; canrenone partially counterbalances the very rapid cell Na+ gain (and K+ depletion) provoked by high ouabain concentrations, and canrenone reverses the secondary effects of ouabain on the Na+,K+-cotransport system and Ca+-dependent, K+-channels. It appears therefore that canrenone may partially reverse the disturbances of cation handling induced by high concentrations of ouabain in macrophages and vascular smooth muscle cells.