Pindolol does not act only on 5-HT1A receptors in augmenting antidepressant activity in the mouse forced swimming test

Hypocapnia produces cerebral vasoconstriction. The mechanisms involved in hypocapnia-induced elevation of vascular smooth muscle tone remain unclear. We addressed the hypothesis that, in cerebrovascular smooth muscle, increases in extracellular pH (pHo) cause increases in Ins(1,4,5)P3 and cytosolic calcium ([Ca2+]c). Superfused primary cultures of piglet cerebral microvascular smooth muscle cells were exposed to artificial CSF (aCSF) of control (pHo 7. 4, PCO2 36 mm Hg), metabolic alkalosis (pHo 7.7, PCO2 36 mm Hg), or respiratory alkalosis (pHo 7.7, PCO2 19 mm Hg). Intracellular pH (pHi) and [Ca2+]c were measured, using BCECF and fura-2, respectively, with dual wavelength spectroscopy. Ins(1,4,5)P3 was determined by a protein binding assay. Both metabolic and respiratory acidosis treatments increased pHi from the control value of about 7.2 to 7.35. Metabolic and respiratory alkalosis increased Ins(1,4,5)P3, as we showed previously. Metabolic and respiratory alkalosis increased [Ca2+]c about 80% and 110%, respectively. Neither Ins(1,4,5)P3 nor [Ca2+]c increased in cells treated with aCSF that produced control pHo with increased pHi (7.3). In contrast, when pHo increased (7.7), but pHi was maintained at control (7.2), Ins(1,4,5)P3 increased from 123 pmol/well to 307 pmol/well and [Ca2+]c increased 46%. However, the increase of [Ca2+]c was less than with either respiratory or metabolic alkalosis. Thus, hypocapnia-induced cerebral vasoconstriction could involve production of Ins(1,4,5)P3 with resultant elevation in [Ca2+]c. While the Ins(1,4,5)P3 signal appears to be dependent on an increase in extracellular pH, a role for intracellular pH cannot be completely excluded.     

Effects of the nitric oxide donor sodium nitroprusside on intracellular pH and contraction in hypertrophied myocytes

BACKGROUND: We compared the effects of the nitric oxide donor sodium nitroprusside (SNP) on intracellular pH (pHi), intracellular calcium concentration ([Ca2+]i) transients, and cell contraction in hypertrophied adult ventricular myocytes from aortic-banded rats and age-matched controls. METHODS AND RESULTS: pHi was measured in individual myocytes with SNARF-1, and [Ca2+]i transients were measured with indo 1 simultaneously with cell motion. Experiments were performed at 37 degrees C in myocytes paced at 0.5 Hz in HEPES-buffered solution (extracellular pH = 7.40). At baseline, calibrated pHi, diastolic and systolic [Ca2+]i values, and the amplitude of cell contraction were similar in hypertrophied and control myocytes. Exposure of the control myocytes to 10(-6) mol/L SNP caused a decrease in the amplitude of cell contraction (72 +/- 7% of baseline, P < .05) that was associated with a decrease in pHi (-0.10 +/- 0.03 U, P < .05) with no change in peak systolic [Ca2+]i. In contrast, in the hypertrophied myocytes exposure to SNP did not decrease the amplitude of cell contraction or cause intracellular acidification (-0.01 +/- 0.01 U, NS). The cGMP analogue 8-bromo-cGMP depressed cell shortening and pHi in the control myocytes but failed to modify cell contraction or pHi in the hypertrophied cells. To examine the effects of SNP on Na(+)-H+ exchange during recovery from intracellular acidosis, cells were exposed to a pulse and washout of NH4Cl. SNP significantly depressed the rate of recovery from intracellular acidosis in the control cells compared with the rate in hypertrophied cells. CONCLUSIONS: SNP and 8-bromo-cGMP cause a negative inotropic effect and depress the rate of recovery from intracellular acidification that is mediated by Na(+)-H+ exchange in normal adult rat myocytes. In contrast, SNP and 8-bromo-cGMP do not modify cell contraction or pHi in hypertrophied myocytes.     

Ischemic preconditioning: effects on pH, Na and Ca in newborn rabbit hearts during Ischemia/Reperfusion

In adult hearts, ischemic preconditioning (PC) has been shown to decrease ischemia-induced changes in intracellular pH (pHi) and [Ca] ([Ca]i) and decrease associated injury. These results are consistent with the interpretation that PC decreases the stimulus for Na uptake via Na/H exchange, thereby decreasing intracellular Na (Nai) accumulation, and thus decreasing the change in force driving Na/Ca exchange, which otherwise contributes to ischemia-induced increases in [Ca]i. Given documented age-related differences in myocardial responses to ischemia, we tested the hypothesis that in newborn hearts, PC will diminish intracellular [H], Nai, and [Ca]i during ischemia/reperfusion. NMR was used to measure pHi, Nai, [Ca]i, ATP, and PCr in isolated newborn (4-7 days) rabbit hearts Langendorff-perfused with Krebs-Henseleit solution equilibrated with 95% O2/5% CO2 at 36+/-1 degrees C. Control hearts were perfused 30 min before initiating 40 min global ischemia followed by 40 min reperfusion. PC hearts were treated the same except four 5-min intervals of ischemia each followed by 10 min of perfusion which preceded global ischemia. At end ischemia, pHi was higher in PC than control hearts (6.31+/-0.03 v 5.83+/-0.05; P<0.05). Similarly, PC diminished Nai-accumulation during ischemia and reperfusion (P<0.05). Control Nai rose from 16.2+/-2.6 to 108.8+/-10.3 (mEq/kg dry weight) and recovered to 55.2+/-10.1 and the corresponding values for PC hearts were 25.6+/-6.2, 70.0+/-7.9 and 21.9+/-5.2. PC also improved [Ca]i recovery during reperfusion (P<0.05). Control [Ca]i rose from 418+/-43 to 1100+/-78 (nm/l) and recovered to 773+/-63, whereas in PC hearts the values were 382+/-40, 852+/-136 and 371+/-45, respectively. In addition, PC decreased coronary resistance during reperfusion (P<0.05) as reflected by lower perfusion pressures under constant flow conditions (65.9+/-1.5 v 56. 1+/-4.1 mmHg at end of reperfusion). Finally, PC improved recovery of left-ventricular developed pressure (LVDP-43.8+/-12.0 v 17.2+/-3. 0% of control; P<0.05) and diminished CK release (607+/-245 v 2432+/-639 IU/g dry weight; P<0.05) during reperfusion. The results are consistent with the hypothesis.     

Roles of calcium-activated and voltage-gated delayed rectifier potassium channels in endothelium-dependent vasorelaxation of the rabbit middle cerebral artery

1. The cellular mechanism(s) of action of endothelium-derived vasodilator substances in the rabbit middle cerebral artery (RMCA) were investigated. Specifically, the subtypes of potassium channels involved in the effects of endothelium-derived relaxing factors (EDRFs) in acetylcholine (ACh)-induced endothelium-dependent vasorelaxation in this vessel were systematically compared. 2. In the endothelium-intact RMCA precontracted with histamine (3 microM), ACh induced a concentration-dependent vasorelaxation, which was sensitive to indomethacin (10 microM) or N(G)-nitro-L-arginine (L-NOARG; 100 microM); pD2 values 8.36 vs 7.40 and 6.38, P < 0.01 for both, n = 6 and abolished by a combination of both agents. ACh caused relaxation in the presence of high K+ PSS (40 mM KCl), which was not affected by indomethacin, but abolished by L-NOARG and a combination of indomethacin and L-NOARG. 3. In the presence of indomethacin, relaxation to ACh in the endothelium-intact RMCA precontracted with histamine was unaffected by either glibenclamide (10 microM), an ATP-sensitive K+ channel (K[ATP]) blocker, 4-aminopyridine (4-AP, 1 mM) or dendrotoxin (DTX, 0.1 microM), delayed rectifier K channel (Kv) blockers. However, relaxation responses to ACh were significantly inhibited by either LY83583 (10 microM) and 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 10 microM), guanylyl cyclase inhibitors, or charybdotoxin (CTX; 0.1 microM), iberiotoxin (ITX, 0.1 microM) and apamin (APA, 0.1 microM), large conductance Ca2+-activated K+ channels (BK[Ca]) blocker and small conductance Ca2+-activated K+ channel (SK[Ca]) blocker, respectively. 4. In the presence of L-NOARG, relaxation to ACh was unaffected by glibenclamide or the cytochrome P450 mono-oxygenase inhibitor, clotrimazole (1 microM), but was significantly inhibited by either 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536, 10 microM) and 2',3'-dideoxyadenosine (2',3'-DDA, 30 microM), adenylyl cyclase inhibitors, or 4-AP, DTX, CTX, ITX and APA. 5. In the endothelium-denuded RMCA precontracted with histamine, authentic NO-induced relaxation was unaffected by glibenclamide, 4-AP and DTX, but significantly reduced by ODQ, ITX and APA. Authentic prostaglandin I2 (PGI2)-induced relaxation was unaffected by glibenclamide, but significantly reduced by 2',3'-DDA, 4-AP, DTX, ITX and APA. Forskolin-induced relaxation was significantly inhibited by high K+, CTX and 4-AP. 6. These results indicate that: (1) in the RMCA the EDRFs released by ACh are NO and a prostanoid (presumably PGI2), and there is no evidence for the release of a non-NO/PGI2 endothelium-derived hyperpolarizing factor (EDHF), (2) K(Ca) channels are involved in NO-mediated relaxation of the RMCA but both K(Ca) and Kv channels are involved in PGI2-mediated relaxation.     

Effect of intracellular acidity and ionomycin on apoptosis in HL-60 cells

The aim was to investigate in detail the influence of intracellular pH (pHi) and intracellular Ca2+ concentration ([Ca2+]i) on apoptosis in HL-60 human promyelocytic leukaemia cells. The pHi was controlled by changing the pH of media as well as by interfering with the pHi regulatory mechanisms with 3-amino-6-chloro-5-(1-homopiperidyl)-N-(diaminomethylene) pyrazincarboxamide (HMA; an inhibitor of Na+/H+ antiport), 4-diiosothiocyanatostilbene-2,2'disulfonic acid, (DIDS; an inhibitor of Na(+)-dependent HCO3-/Cl- exchange) and nigericin (a K+ ionophore). The [Ca2+]i was increased with ionomycin, a Ca2+ ionophore. The apoptosis of HL-60 cells was measured with conventional agarose gel electrophoresis for DNA fragmentation and also with the release of 3H from 3H-thymidine-labelled DNA. Based on the magnitude of DNA fragmentation and 3H release at different pHi, it was shown that apoptosis occurred in HL-60 cells when the pHi was lowered from normal pHi of 7.4 to about 7.2-6.7 with a peak increase at pHi 6.8-6.9. Addition of 4 microM ionomycin to RPMI 1640 medium, which contained 615 microM Ca2+, elevated the apoptosis in the cells. Such an increase in apoptosis by ionomycin in HL-60 cells appeared to result from both an increase in [Ca2+]i and from a decline in pHi. The results indicate that the acidic intratumour environment may greatly affect the response of neoplastic tissues to hyperthermia, radiation and chemotherapeutic drugs which cause apoptosis.     

Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pHi) and intracellular Ca2+ activity ([Ca2+]i) of HT29 cells was investigated microspectrofluorimetrically using pH- and Ca2+- sensitive fluorescent indicators, [i.e. 2', 7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (Vm) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pKa value. Trimethylamine (20 mmol/l) increased pHi by 0.78 +/- 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pHi and a slow and incomplete recovery: pHi stayed 0.26 +/- 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pHi. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca2+]i transient which reached a peak value within 10-25 s and then slowly declined to a [Ca2+]i plateau. The initial Delta[Ca2+]i induced by trimethylamine (20 mmol/l) was 160 +/- 15 nmol/l (n = 17). The [Ca2+]i peak was independent of the Ca2+ activity in the bath solution, but the [Ca2+]i plateau was significantly lower under Ca2+-free conditions and could be immediately interrupted by application of CO2 (10%; n = 6), a manoeuvre to acidify pHi in HT29 cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca2+ stores completely abolished this [Ca2+]i transient. Tetramethylamine led to higher [Ca2+]i changes than the other amines tested and only this transient could be completely blocked by atropine (10(-6) mol/l). Trimethylamine (20 mmol/l) hyperpolarized Vm by 22.5 +/- 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 +/- 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pHi changes, release Ca2+ from intracellular, inositol-1,4, 5-trisphosphate-sensitive Ca2+ stores and increase Ca2+ influx into HT29 cells. The latter may be related to both the store depletion and the hyperpolarization.     

Properties of a novel pH-dependent Ca2+ permeation pathway present in male germ cells with possible roles in spermatogenesis and mature sperm function

Rises of intracellular Ca2+ ([Ca2+]i) are key signals for cell division, differentiation, and maturation. Similarly, they are likely to be important for the unique processes of meiosis and spermatogenesis, carried out exclusively by male germ cells. In addition, elevations of [Ca2+]i and intracellular pH (pHi) in mature sperm trigger at least two events obligatory for fertilization: capacitation and acrosome reaction. Evidence implicates the activity of Ca2+ channels modulated by pHi in the origin of these Ca2+ elevations, but their nature remains unexplored, in part because work in individual spermatozoa are hampered by formidable experimental difficulties. Recently, late spermatogenic cells have emerged as a model system for studying aspects relevant for sperm physiology, such as plasmalemmal ion fluxes. Here we describe the first study on the influence of controlled intracellular alkalinization on [Ca2+]i on identified spermatogenic cells from mouse adult testes. In BCECF [(2',7')-bis(carboxymethyl)- (5, 6)-carboxyfluorescein]-AM-loaded spermatogenic cells, a brief (30-60 s) application of 25 mM NH4Cl increased pHi by approximately 1.3 U from a resting pHi approximately 6.65. A steady pHi plateau was maintained during NH4Cl application, with little or no rebound acidification. In fura-2-AM-loaded cells, alkalinization induced a biphasic response composed of an initial [Ca2+]i drop followed by a two- to threefold rise. Maneuvers that inhibit either Ca2+ influx or intracellular Ca2+ release demonstrated that the majority of the Ca2+ rise results from plasma membrane Ca2+ influx, although a small component likely to result from intracellular Ca2+ release was occasionally observed. Ca2+ transients potentiated with repeated NH4Cl applications, gradually obliterating the initial [Ca2+]i drop. The pH-sensitive Ca2+ permeation pathway allows the passage of other divalents (Sr2+, Ba2+, and Mn2+) and is blocked by inorganic Ca2+ channel blockers (Ni2+ and Cd2+), but not by the organic blocker nifedipine. The magnitude of these Ca2+ transients increased as maturation advanced, with the largest responses being recorded in testicular sperm. By extrapolation, these findings suggest that the pH-dependent Ca2+ influx pathway could play significant roles in mature sperm physiology. Its pharmacology and ion selectivity suggests that it corresponds to an ion channel different from the voltage-gated T-type Ca2+ channel also present in spermatogenic cells. We postulate that the Ca2+ permeation pathway regulated by pHi, if present in mature sperm, may be responsible for the dihydropyridine-insensitive Ca2+ influx required for initiating the acrosome reaction and perhaps other important sperm functions.     

Changes in thyroid state affect pHi and Nai+ homeostasis in rat ventricular myocytes

We have tested the hypothesis that thyroid state may influence both the flow of cellular Ca2+ and the myofilament response to Ca2+ by effects on intracellular pH (pHi) and Na+ (Nai+). Single cardiac myocytes isolated from hypothyroid, euthyroid and hyperthyroid animals were loaded with fura-2/AM (Cai2+ probe), BCECF/AM (pHi probe) or SBFI/AM (Nai+ probe). Compared with hypothyroid animals, myocytes isolated from hyperthyroid rat hearts demonstrated a significant: (1) increase in extent of shortening; (2) decrease in the time to peak contraction; (3) increase in the peak amplitude of the fura-2 fluorescence ratio; (4) decrease in pHi (DeltapHi=0. 19+/-0.05); and (5) increase in Nai+ (DeltaNai+=2.88+/-0.55 mM). We have also compared pHi in Langendorff perfused hypo- and hyperthyroid rat hearts using NMR. We have found that hyperthyroid hearts are 0.15+/-0.03 pH units more acidic than hypothyroid hearts. Analysis of mRNA levels demonstrated that hyperthyroidism increased expression of both the Na+/Ca2+ exchanger and Na+/H+ antiporter, and decreased expression of Na+ channel mRNAs. These changes appear partially responsible for the observed changes in Nai+ and pHi. Our results provide the first evidence that changes in cardiac contractility associated with altered thyroid state not only involve effects on Ca2+, but may also involve changes in the response of the myofilaments to Cai2+mediated by altered pHi and Nai+.     

Effects of pHi on isometric force and [Ca2+]i in porcine coronary artery smooth muscle

During ischemia or hypoxia, alterations in pHi may play a significant role in alteration of vessel wall function. We studied the effects of altering pHi on isometric force and [Ca2+]i in porcine coronary artery. pHi was altered at constant pHo by use of NH4Cl and measured with the fluorescent dye BCECF. [Ca2+]i was monitored with fura 2 and ratiometric fluorescence measurements. Addition of NH4Cl elicited a concentration-dependent (2 to 30 mmol/L) sustained increase in isometric force in unstimulated tissues. In tissues stimulated with KCl (29 mmol/L) or U46619 (1 mumol/L), addition of NH4Cl elicited a rapid but transient decrease followed by a sustained increase in force above the initial stimulated levels. Removal of NH4Cl was associated with a transient decrease and increase followed by a prolonged depression of force and slow recovery to initial levels. Addition of NH4Cl elicited a rapid monotonic increase in pHi and then a slow recovery toward initial levels; washout of NH4Cl led to a rapid acidification followed by recovery. In contrast to the steady state effects of NH4Cl on force, its effects on [Ca2+i were in the opposite direction. During the sustained increase in force elicited by NH4Cl alkalinization, [Ca2+]i was substantially decreased, whereas when force was depressed during the acidification elicited by NH4Cl washout, [Ca2+i was increased to values observed before addition of NH4Cl. The initial transients in force elicited by NH4Cl addition or washout were also associated with opposite changes in [Ca2+]i. Thus, the effects on force of the NH4Cl-induced changes in pHi are associated with changes in the Ca2+ sensitivity of the contractile apparatus rather than mediated through changes in [Ca2+]i.     

Glibenclamide-sensitive mechanism is involved in helodermin-produced vasodilatation in rat mesenteric artery

Helodermin-caused vascular relaxation was simultaneously measured with intracellular Ca2+ concentration ([Ca2+]i) in rat mesenteric artery. Helodermin caused concentration-dependent relaxation in the mesenteric artery preconstricted with norepinephrine (NE). Helodermin-caused relaxation was accompanied by decrease in [Ca2+]i, D-cis-Diltiazem, a Ca2+ channel blocker, also lowered the [Ca2+]i and tension increased by NE. However, helodermin relaxed the artery more efficiently than D-cis-diltiazem, suggesting that the peptide decreased myofilament Ca2+ sensitivity. The vascular relaxation and the corresponding decrease in [Ca2+]i induced by helodermin were partly, but significantly attenuated by glibenclamide. Helodermin-induced vascular responses were mimicked by vasoactive intestinal polypeptide (VIP) or forskolin. Furthermore, helodermin increased cAMP contents in the mesenteric artery. These findings show that vasodilatation induced by helodermin is attributable to lowered [Ca2+]i of arterial smooth muscle partly through the activation of glibenclamide-sensitive K+ channels, and to decrease in the myofilament Ca2+ sensitivity. The increase in the cellular cAMP content probably plays a key role in the peptide-induced vasorelaxation.     

Dose-dependent effects of chloroquine on secretory granule formation in the melanotroph

Intracellular calcium ([Ca2+]i) and hydrogen ion concentrations (pHi) are important regulators of cell function. Those ions also may interact and it is important, therefore, to measure their concentrations simultaneously. In the present studies we used a system developed for that purpose, a fluorescent emission ratio technique for simultaneous analysis of calcium (Indo-1) and pH (SNARF-1) in single cells at video rates, and determined if arginine vasopressin (AVP, 12.5 mumol/l) evoked [Ca2+]i and pHi signals interact in MDCK cells. We also employed a simple system for analysing the side specific (basolateral or apical) application of agonist to polarized cell layers on permeable membranes. AVP is found to evoke simultaneous changes in both pHi and [Ca2+]i. Basolateral application induced transient acidification, followed by partial recovery, and a [Ca2+]i transient with kinetic pattern similar to that of the pHi. Apical application also caused a mirror image pHi and [Ca2+]i pattern but of smaller magnitude (no peak). Selective removal of extracellular calcium ([Ca2+]e) or sodium ([Na+]e) dissociated the pHi and [Ca2+]i responses in both cases. Na+e removal abolished the pHi changes, but not the [Ca2+]i transients. [Ca2+]e removal abolished the [Ca2+]i changes and reduced, but did not abolish, the pHi responses. Thus, AVP induces pHi changes which are modified by calcium while calcium signalling is not modified by changes in pHi.     

Rapid kinetics of myo-inositol trisphosphate binding and dissociation in cerebellar microsomes

Using sheep cerebellum microsomes adsorbed on a filter, we measured the kinetics of [3H]inositol 1,4,5-trisphosphate (InsP3) binding and dissociation on the subsecond time scale during rapid perfusion of the filter with [3H]InsP3-containing or InsP3-free media. At 20 degrees C and pH 7.1, in a cytosol-like medium containing MgCl2, the half-time for InsP3 dissociation was as short as 125 ms. The receptor behaved as a simple target for binding of its ligand, with the rate constant for InsP3 binding increasing linearly with InsP3 concentration. Various modulators of InsP3 binding (KCl, NaCl, pH, Mg2+, and Ca2+) were found to affect the receptor's apparent affinity for InsP3 mainly by altering the rate constant for [3H]InsP3 dissociation. ATP (but not InsP3) also accelerated [3H]InsP3 dissociation. In contrast to these modulators, luminal Ca2+ was found to have no effect on the amount of microsome-bound [3H]InsP3.     

Intracellular pH buffering shapes activity-dependent Ca2+ dynamics in dendrites of CA1 interneurons

Voltage-gated calcium (Ca) channels are highly sensitive to cytosolic H+, and Ca2+ influx through these channels triggers an activity-dependent fall in intracellular pH (pHi). In principle, this acidosis could act as a negative feedback signal that restricts excessive Ca2+ influx. To examine this possibility, whole cell current-clamp recordings were taken from rat hippocampal interneurons, and dendritic Ca2+ transients were monitored fluorometrically during spike trains evoked by brief depolarizing pulses. In cells dialyzed with elevated internal pH buffering (high beta), trains of >15 action potentials (Aps) provoked a significantly larger Ca2+ transient. Voltage-clamp analysis of whole cell Ca currents revealed that differences in cytosolic pH buffering per se did not alter baseline Ca channel function, although deliberate internal acidification by 0.3 pH units blunted Ca currents by approximately 20%. APs always broadened during a spike train, yet this broadening was significantly greater in high beta cells during rapid but not slow firing rates. This effect of internal beta on spike repolarization could be blocked by cadmium. High beta also 1) enhanced the slow afterhyperpolarization (sAHP) seen after a spike train and 2) accelerated the decay of an early component of the sAHP that closely matched a sAHP conductance that could be blocked by apamin. Both of these effects on the sAHP could be detected at high but not low firing rates. These data suggest that activity-dependent pHi shifts can blunt voltage-gated Ca2+ influx and retard submembrane Ca2+ clearance, suggesting a novel feedback mechanism by which Ca2+ signals are shaped and coupled to the level of cell activity.     

Clinical utility of Lens culinaris agglutinin-reactive alpha-fetoprotein in small hepatocellular carcinoma: special reference to imaging diagnosis

We sought to determine whether insulin/insulin-like growth factor-1 (IGF-1) and an insulin-sensitizing agent, troglitazone, have additive vasodilatory effects and the possible involvement of intracellular Ca2+ ([Ca2+]i) and/or glucose utilization in these effects. Contractile responses to norepinephrine (NE) and potassium chloride (KCl), as well as relaxation to endothelium-dependent (acetylcholine [Ach]) and -independent (sodium nitroprusside [NaNP]) agents, were examined in rat tail artery rings in the presence of insulin/IGF-1 and/or troglitazone. Endothelium-intact tail artery rings stretched to 1 g tension were preincubated with troglitazone (3 micromol/L) and/or insulin/IGF-1 (100 nmol/L) prior to addition of graded doses of NE and KCI. A 90-minute exposure to troglitazone attenuated the maximal contraction to graded doses of NE and KCI (P<.0001). Incubation in glucose-free medium decreased the responses only to NE; troglitazone further attenuated the NE-induced contraction (P = .001). In submaximally precontracted endothelium-intact rings, troglitazone increased the relaxation both to NaNP (P<.0001) and to Ach (P = .001). Contraction experiments in depolarizing KCI (25 mmol/L) or Ca2+ -free buffer showed that troglitazone and insulin have a similar Ca2+ dependency. In conclusion, troglitazone, like insulin/IGF-1, attenuates responses to vasoactive agonists through a Ca2+ -dependent mechanism that may require the presence of glucose but is independent of insulin action and nitric oxide (NO) production.     

High PCO does not alter pHi, but raises [Ca2+]i in cultured rat carotid body glomus cells in the absence and presence of CdC12

We measured the effect of high PCO (500-550 Torr) on the pHi and [Ca2+]i in cultured glomus cells of adult rat carotid body (CB) as a test of the two models currently proposed for the mechanism of CB chemoreception. The metabolic model postulates that the rise in glomus cell [Ca2+]i, the initiating reaction in the signalling pathway leading to chemosensory neural discharge, is due to [Ca2+] release from intracellular Ca2+ stores. The membrane potential model postulates that the rise in [Ca2+]i comes from influx of extracellular Ca2+ through voltage-dependent Ca2+ channels (VDCC) of the L-type. High PCO did not change pHi at PO2 of 120-135 Torr, showing that CO-induced changes in [Ca2+]i are not due to changes in pHi. High PCO caused a highly significant rise in [Ca2+]i from 90+/-12 nM to 675+/-65 nM, both in the absence and in the presence of 200 microM CdCl2, a potent blocker of L-type VDCCs. This result is fully consistent with release of Ca2+ from glomus cell intracellular stores according to metabolic model, but inconsistent with influx of extracellular Ca2+ through VDCCs according to the membrane potential model.     

In vivo uptake of [3H]nimodipine in focal cerebral ischemia: modulation by hyperglycemia

Cell membrane depolarization and tissue acidosis occur rapidly in severely ischemic brain. Preischemic hyperglycemia is recognized to increase ischemic tissue acidosis and the present studies were undertaken to correlate depolarization and tissue acidosis during acute focal cerebral ischemia and hyperglycemia. We used a dual-label autoradiography method to simultaneously measure the in vivo distribution of [3H]nimodipine and [14C]DMO (5,5-dimethyl-2,4-oxazolidinedione) in brain to identify regions of ischemic depolarization and measure regional net tissue pH. Regional cerebral blood flow (CBF) was measured in separate studies. Measurements were made 30 minutes after combined middle cerebral artery and ipsilateral common carotid artery occlusion in normoglycemic and hyperglycemic rats. Tissue pH in the ischemic cortex was depressed to 6.76 +/- 0.11 in normoglycemic rats (n = 12) and 6.57 +/- 0.13 in hyperglycemic rats (n = 12), with significantly greater acidosis in the hyperglycemic group (P < 0.001). In contrast the ratio of [3H]nimodipine uptake in the ischemic cortex relative to the contralateral nonischemic cortex was significantly greater in normoglycemic (1.83 +/- 0.45) than hyperglycemic (1.40 +/- 0.50) rats (P < 0.05). Within this region of ischemic cortex CBF was 31 +/- 22 mL/100 g in normoglycemic rats (n = 8) and 33 +/- 22 mL/100 g/min in hyperglycemic rats (n = 9). Cerebral blood flow did not differ between these two groups in any region. Thus hyperglycemia reduced the extent of ischemic depolarization within the cortex during the first 30 minutes of focal cerebral ischemia. This effect may be related to the increased tissue acidosis or to other factors that may lessen calcium influx and preserve cellular energy stores in the ischemic cortex of the hyperglycemic rats.     

Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators

Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in furaptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50-100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in vitro calibration parameters.     

Endothelin-A receptors mediate vascular smooth-muscle response to moderate acidosis in the canine tibial nutrient artery

Perfusate pH may influence the tone of vascular smooth muscle by affecting the release of endothelium-derived vasoactive factors or by directly modulating function of the smooth muscle. This study was designed to investigate the role of endothelium-derived factors on acidosis-induced responses of isolated canine tibial nutrient artery suspended in an organ chamber for the measurement of isometric contractile force. To investigate the specific role of the endothelium in half the rings, the endothelium was removed mechanically. Concentration-response curves to KCl were obtained in the absence or presence of inhibition of two important endothelium-derived relaxing factors, nitric oxide and prostacyclin, and an inhibitor of receptors for the endothelium-derived contracting factor, endothelin-1. Acidification of the perfusate from pH 7.45 to 7.0 significantly attenuated the contractions to KCl in arterial rings with endothelium (the mean of the effective concentration causing 50% of the maximal response for KCl at pH 7.45 and 7.0 was 12.31 +/- 0.40 nM and 14.60 +/- 0.55 nM, respectively). This difference was abolished by mechanical removal of the endothelium. In rings with endothelium, inhibition of nitric oxide or prostacyclin did not abolish the attenuation of KCl-induced contractions occurring with acidosis (the mean of the effective concentration causing 50% of the maximal response for KCl at pH 7.45 and 7.0 was 11.18 +/- 0.60 nM and 13.60 +/- 0.60 nM, respectively). Inhibition of endothelin-A receptors did not alter contractions to KCl at pH 7.45. However, the acidosis-induced attenuation of contractions with KCl was abolished by the endothelin-A-receptor antagonist BQ-123 (the mean of the effective concentration causing 50% of the maximal response at pH 7.45 and 7.0 was 13.8 +/- 1.34 nM and 13.2 +/- 1.34 nM, respectively). These results suggest that acidosis-induced relaxation of canine tibial nutrient artery is endothelium dependent and that activation of endothelin-A receptors during acidosis is coupled to a release of an endothelium-derived relaxing factor.     

Ethanol acutely decreases calcium transients in cultured human myotubes

Ethanol consumption frequently leads to a number of skeletal muscle disorders, including acute and chronic alcoholic myopathy. Ethanol has been found to interfere with signal transduction mechanisms in cardiac and smooth muscle cells. We studied the effects of ethanol on the intracellular calcium ([Ca2+]i) transients responsible for excitation-contraction coupling in human myotubes from chronic alcoholic patients and healthy controls. Cultured myotubes were loaded with the fluorescent Ca2+ indicator fura-2 and evaluated on a single-cell basis. Following electrical stimulation, ethanol caused a significant reversible dose-dependent reduction in [Ca2+]i transient amplitude, achieving a mean decrease of 36+/-5% at 300 mM ethanol (p < 0.01), without modifying the basal [Ca2+]i. This acute effect of ethanol was similar in myotubes obtained from chronic alcoholics and controls. Similarly, ethanol caused a dose-dependent reduction of [Ca2+]i transient amplitude in control samples when depolarization was elicited by 100 mM KCl (p < 0.01). Several potential mechanisms of ethanol action were studied in control muscle samples. Sarcolemmal Ca2+ entry was measured indirectly by monitoring Mn2+-quenching of intracellular fura-2 via the nitrendipine-sensitive Ca2+ channels during electrical pacing. Ethanol at doses of 100 mM and greater caused a dose-dependent reduction in the rate of quench (p < 0.01). In addition, the intracellular pool of Ca2+ releasable by caffeine was found to be reduced at 300 mM ethanol (p < 0.05). We conclude that ethanol reduces the [Ca2+]i transients underlying excitation-contraction coupling in human myotubes, and that this occurs to a similar extent in cells obtained from chronic alcoholics and controls. This acute effect of ethanol was primarily due to an inhibitory effect of ethanol on sarcolemmal Ca2+ influx via voltage-operated Ca2+ channels, although there may also be an effect on the Ca2+ sarcoplasmic reticulum loading state.     

A comparative evaluation of the effects of multiple vasodilators on human internal mammary artery

BACKGROUND: Vasospasm of arterial grafts represents an unpredictable complication of coronary artery surgery and may compromise myocardial revascularization, and treatment is based on empirical therapy with nitroglycerin. Because of the potential for tolerance to nitroglycerin to occur, the authors studied different vasodilators acting through separate pathways on segments of human internal mammary artery. METHODS: Isolated vascular rings were precontracted with norepinephrine (1 microM), KCl, or the thromboxane A2 analogue (U46619, 10 nm). Nitroglycerin (a nitrovasodilator), milrinone (a type III phosphodiesterase inhibitor), papaverine (a phosphodiesterase inhibitor), prostaglandin E1, and isradipine (a dihydropyridine calcium channel blocker) were added in a cumulative fashion. RESULTS: The analysis of the concentration-response curves showed that vasodilators induced 90-100% relaxation of the constricted segments with norepinephrine or the thromboxane A2 analogue, except prostaglandin E1, which produced 73% relaxation at maximal concentrations. The effective concentrations of vasodilator agent that caused 50% relaxation for nitroglycerin and milrinone were within the range of the reported therapeutic concentrations in plasma. Isradipine was also effective at reversing receptor-mediated contraction (maximal relaxation=100% in internal mammary artery contracted with norepinephrine; maximal relaxation=0% in internal mammary artery contracted with the thromboxane A2 analogue). CONCLUSIONS: Vasodilator drugs acting through multiple pathways are effective at reversing in vitro vasoconstriction.