Exogenous detection of 13C-glucose metabolism in tumor and diet-induced obesity models.

Metabolic rewiring is a hallmark feature prevalent in cancer cells as well as insulin resistance (IR) associated with diet-induced obesity (DIO). For instance, tumor metabolism shifts towards an enhanced glycolytic state even under aerobic conditions. In contrast, DIO triggers lipid-induced IR by impairing insulin signaling and reducing insulin-stimulated glucose uptake. Based on physiological differences in systemic metabolism, we used a breath analysis approach to discriminate between different pathological states using glucose oxidation as a readout. We assessed glucose utilization in lung cancer-induced cachexia and DIO mouse models using a U-13C glucose tracer and stable isotope sensors integrated into an indirect calorimetry system. Our data showed increased 13CO2 expired by tumor-bearing (TB) mice and a reduction in exhaled 13CO2 in the DIO model. Taken together, our findings illustrate high glucose uptake and consumption in TB animals and decreased glucose uptake and oxidation in obese mice with an IR phenotype. Our work has important translational implications for the utility of stable isotopes in breath-based detection of glucose homeostasis in models of lung cancer progression and DIO.

INSPIRE: A European training network to foster research and training in cardiovascular safety pharmacology.

Safety pharmacology is an essential part of drug development aiming to identify, evaluate and investigate undesirable pharmacodynamic properties of a drug primarily prior to clinical trials. In particular, cardiovascular adverse drug reactions (ADR) have halted many drug development programs. Safety pharmacology has successfully implemented a screening strategy to detect cardiovascular liabilities, but there is room for further refinement. In this setting, we present the INSPIRE project, a European Training Network in safety pharmacology for Early Stage Researchers (ESRs), funded by the European Commission's H2020-MSCA-ITN programme. INSPIRE has recruited 15 ESR fellows that will conduct an individual PhD-research project for a period of 36 months. INSPIRE aims to be complementary to ongoing research initiatives. With this as a goal, an inventory of collaborative research initiatives in safety pharmacology was created and the ESR projects have been designed to be complementary to this roadmap. Overall, INSPIRE aims to improve cardiovascular safety evaluation, either by investigating technological innovations or by adding mechanistic insight in emerging safety concerns, as observed in the field of cardio-oncology. Finally, in addition to its hands-on research pillar, INSPIRE will organize a number of summer schools and workshops that will be open to the wider community as well. In summary, INSPIRE aims to foster both research and training in safety pharmacology and hopes to inspire the future generation of safety scientists.

Contribution of frequency-augmented inward Ca2+ current to myocardial contractility.

The sarcoplasmic reticular Ca2+ pump (SERCA) is thought to be the primary determinant of heart rate-dependent increases in myocardial contractile [Ca2+]i and force (force-frequency relationship (FFR)), an important mechanism to increase cardiac output. This report demonstrates a rate-dependent role for inward Ca2+ current (ICa) in the human and rat FFR. Human action potential plateau height increased linearly with contractility when heart rate increased in vivo, as measured by monophasic action potential catheter and echocardiography. Rat rate-dependent developed force and cytosolic [Ca2+]i transients were quantified in isolated left ventricular papillary muscles, and ICa and action potential duration in cardiomyocytes. ICa and SERCA measurements better reflected [Ca2+]i and force transients than SERCA activity alone. These data support a direct and (or) indirect contribution to myocardial contractility by ICa at heart rates from approximately 1 to 3-4 Hz (60 to 180-240 bpm) in tandem with SERCA to sustain the typical 'bell shape' of the FFR across species.

Myogenic tone and reactivity of cerebral arteries in type II diabetic BBZDR/Wor rat.

BBZDR/Wor rat is a new model of type II diabetes with spontaneous obesity and clinical characteristics close to human diabetes. In this study the time-course of cerebroarterial dysfunction was characterized. Posterior cerebral arteries from BBZDR/Wor rats and their age-matched lean controls were pressurized to 70 mm Hg in an arteriograph. Effects of intraluminal pressure and different pharmacological agents on myogenic tone were evaluated. Pressure-myogenic tone curves in diabetic arteries were similar to that in non-diabetic arteries at pre-diabetic age, showed leftward shift at 4 weeks and were significantly different with higher myogenic tone at 5 and 8 months of diabetes. Age-dependent decrease in myogenic tone was observed in non-diabetic arteries. Dilation to histamine was similar to that in non-diabetic arteries at pre-diabetic and at 4 weeks but significantly reduced at 5 and 8 months of diabetes. Bradykinin-mediated dilation was significantly reduced in early and chronic diabetes, whereas (+/-)-S-nitroso-N-acetylpenicillamine (SNAP)-mediated dilation was decreased modestly at 8 months of diabetes. Sensitivity and constriction to 5-hydroxytryptamine were increased in early and chronic diabetes. Responses to bradykinin and 5-hydroxytryptamine were decreased and increased, respectively. Myogenic tone was significantly less sensitive to (lower pIC(50)) U-73122 than normal arteries at 4 weeks and 8 months of diabetes suggesting an increased activation of phospholipase C (PLC). This study shows that pressure-mediated autoregulation of cerebral arteries in type II diabetes operates at higher resistance. Endothelium-dependent dilation was decreased with chronic diabetes with increased sensitivity to constrictor agonist. Endothelium-independent dilation was modestly affected. Arterial hyper-reactivity to pressure and constrictor agonist were likely due to increased PLC activation.

KCNMB1 genotype influences response to verapamil SR and adverse outcomes in the INternational VErapamil SR/Trandolapril STudy (INVEST).

OBJECTIVES: We sought to determine whether polymorphisms in the large-conductance calcium and voltage-dependent potassium (BK) channel beta1 subunit gene, KCNMB1, are associated with blood pressure response to verapamil SR or adverse outcomes in the GENEtic substudy of the INternational VErapamil SR/trandolapril STudy (INVEST-GENES). BACKGROUND: KCNMB1 is involved in calcium sensitivity and hypertension. The association between variability in KCNMB1 and calcium antagonist response, however, has not been assessed. METHODS: Genetic samples were collected from 5979 patients in INVEST. Blood pressure response to verapamil SR and time to achieve blood pressure control was assessed in relation to Glu65Lys and Val110Leu genotypes. The primary outcome (all cause mortality, nonfatal myocardial infarction or nonfatal stroke) was compared between genotype groups, and interaction with verapamil SR therapy was assessed. RESULTS: Systolic blood pressure response to verapamil SR did not differ by KCNMB1 genotype. Lys65 variant carriers, however, achieved blood pressure control earlier than Glu65Glu individuals [1.47 (interquartile ratio 2.77) versus 2.83 (interquartile ratio 4.17) months, P=0.01] and were less likely to require multiple drugs at the time of blood pressure control (adjusted odds ratio 0.43, 95% confidence interval 0.19-0.95). Leu110 variant carriers had a reduced risk of primary outcome (hazard ratio 0.68, 95% confidence interval 0.47-0.998). Subgroup analysis revealed this finding to be more pronounced in verapamil SR-assigned patients (hazard ratio 0.587, 95% confidence interval 0.33-1.04) compared with atenolol-assigned patients (hazard ratio 0.946, 95% confidence interval 0.56-1.59). No difference was seen in the occurrence of the primary outcome compared by codon 65 genotype. CONCLUSIONS: Our findings suggest that KCNMB1 genotype influences responsiveness to verapamil SR and risk of adverse cardiovascular outcomes.

Characteristics of myogenic tone in the rat ophthalmic artery.

The pressure-induced constriction in the rat ophthalmic artery was characterized. Ophthalmic arteries were isolated, cannulated in an arteriograph, and pressurized. Arteries developed 25% constriction at 70 mmHg of intraluminal pressure. Arteries maintained almost similar diameter over the range of pressures 50-210 mmHg, and forced dilatation was observed at pressures >210 mmHg. Denudation of endothelium increased the sensitivity of arteries to pressure-induced constriction, and significantly higher myogenic tone was observed in the pressure range of 10-100 mmHg. Indomethacin and cyclooxygenase-2 inhibition by SC-236 decreased myogenic tone, whereas cyclooxygenase-1 inhibition by SC-560 potentiated myogenic tone in a lower concentration range and decreased at a higher concentration. Pressure-induced constriction was completely blocked by 1 microM nifedipine. Phospholipase C inhibition by 6 microM U-73122 decreased myogenic tone by 39%, whereas PKC inhibitor GF-109203X (3 microM) had no effect. Constriction to phenylephrine was significantly decreased by U-73122 (1 microM) and GF-109203X (3 microM) at an intraluminal pressure of 10 mmHg. Rho-kinase inhibition by Y-27632 (30 microM) and HA-1077 (30 microM) decreased myogenic tone by 75% and 73%, respectively, and 1 microM Y-27632 significantly decreased myogenic tone developed in response to graded increases in pressure. These results suggest that rat ophthalmic artery has an efficient pressure-dependent autoregulatory function that is modulated by endothelium. Contribution of phospholipase C-activation to myogenic tone is minimal, whereas Rho-kinase activation plays a predominant role in the myogenic reactivity in this artery.

Histamine decreases myogenic tone in rat cerebral arteries by H2-receptor-mediated KV channel activation, independent of endothelium and cyclic AMP.

The effect of histamine on the pressure-induced constriction was characterized in rat cerebral arteries and mechanisms were investigated. Rat cerebral arteries were pressurized to 70 mm Hg in an arteriograph and the effect of histamine on myogenic tone was studied. Histamine and amthamine, a selective histamine H(2)-receptor agonist, concentration-dependently decreased myogenic tone, which was unchanged in the absence of endothelium. 2-(2-aminoethyl) pyridine, a selective histamine H(1)-receptor agonist, produced concentration-dependent constriction of arteries that was significantly increased in the absence of endothelium. Imetit, a selective histamine H(3)-receptor agonist, has no effect on myogenic tone. The dilation to histamine was antagonized by tiotidine, a selective antagonist of histamine H(2)-receptor subtype, giving a pK(B) of 7.86 that was not altered in the absence of endothelium. The histamine-mediated dilation was significantly antagonized by NF 449, a reversible inhibitor of Gs-protein activation but was not affected by ODQ and SQ 22536. Dilations to histamine and amthamine were accompanied by a decrease in arterial wall calcium measured by fura-2 ratios. The dilation to histamine was significantly reduced by partial depolarization of smooth muscle by 25 mM KCl (control 91+/-5%, 25 mM KCl 53+/-5%, P<0.002) and was not observed in the presence of strongly depolarizing 60 mM KCl. The histamine dilation was not affected by iberiotoxin, barium chloride and glibenclamide but was strongly antagonized by 4-aminopyridine (0.3 mM) and tetraethylammonium chloride (10 mM) (pEC(50): control: 5.6+/-0.1, 4-aminopyridine: 4.1+/-0.1 (P<0.001); tetraethylammonium chloride: 3.2+/-0.2 (P<0.0001)). These results suggest that histamine-mediated reversal of myogenic tone in rat cerebral arteries is endothelium-independent, mediated by histamine H(2)-receptor subtype with no involvement of guanylyl cyclase or adenylyl cyclase activation and most likely involves activation of K(V) potassium channels.

Muscarinic acetylcholine type-3 receptor desensitization due to chronic exposure to Sjögren's syndrome-associated autoantibodies.

OBJECTIVE: Anti-muscarinic acetylcholine type-3 receptor (anti-M3R) autoantibodies have been shown to inhibit M3R-mediated responses by both in vitro contractility and fura-2 microfluorimetry analyses of intracellular calcium mobilization, suggesting an important role for anti-M3R autoantibody in secretory dysfunction. We investigated whether chronic stimulation of M3R by anti-M3R autoantibodies and/or the agonist pilocarpine results in a general M3R desensitization. METHODS: Carbachol-evoked responses of mouse-bladder smooth muscle strips were measured following exposure to anti-M3R-positive and/or anti-M3R-negative sera from either NOD/Lt mice, a model of SS-like disease, or human patients with primary SS. RESULTS: Bladder smooth muscle strips isolated from NOD/Lt mice with circulating anti-M3R autoantibodies exhibited lower carbachol-evoked responses than smooth muscle strips from anti-M3R autoantibody-negative NOD/Lt mice and control C57BL/6 mice. Repeated pilocarpine injections of NOD mice for 6 days also revealed M3R desensitization in the agonist-evoked contractile assay, whereas age and sex matched C57BL/6 mice injected with pilocarpine for the same period showed a 2-fold higher response. Incubation of smooth muscle strips with sera obtained from patients with primary SS resulted in both stimulated and inhibited responses. CONCLUSION: These results support the hypothesis that chronic stimulation of membrane-bound M3R can result in receptor desensitization, and raise questions about repeated use of pilocarpine by patients positive for anti-M3R autoantibodies.

Myogenic tone and reactivity of rat ophthalmic artery in acute exposure to high glucose and in a type II diabetic model.

PURPOSE: To evaluate the effect of acute exposure to high glucose on myogenic tone and reactivity of the rat ophthalmic artery and to compare the observations with that in ophthalmic artery from a diabetic rat model. METHODS: Ophthalmic arteries from Sprague-Dawley rats were pressurized at 70 mmHg in an arteriograph, and outer diameter was monitored. Myogenic tone was assessed over a range of intraluminal pressures in the presence and absence of high glucose or mannitol. The effects of high glucose on reactivity to carbachol and phenylephrine were determined. Arteries from type II diabetic BBZDR/Wor rats and age-matched control rats were evaluated for myogenic tone and reactivity. RESULTS: Myogenic tone was enhanced by 25 mM, but decreased by 40 mM, glucose and was not affected by mannitol. Constriction to phenylephrine was not affected by 25 mM, but was decreased by 40 mM glucose, and carbachol-mediated dilation was unaffected. Effects of high glucose were not observed in the absence of endothelium. Miconazole, a nonselective inhibitor of cytochrome-P450 enzymes or dihydro-ouabain, an inhibitor of Na+,K+-ATPase blocked the effect of 40 mM but not 25 mM glucose. Arteries from diabetic rats showed decreased myogenic tone compared with control arteries, and this decrease was not observed in the absence of endothelium. CONCLUSIONS: Acute exposure to high glucose has a concentration- and endothelium-dependent effect on the myogenic tone of rat ophthalmic artery. Attenuation of tone by high glucose is probably due to the activation of smooth muscle Na+,K+-ATPase by endothelial cytochrome-P450 metabolite. Pressure-mediated autoregulation in ophthalmic artery in type II diabetic BBZDR/Wor rat operates at lower resistance, probably due to hyperglycemia.

Relative contribution of Rho kinase and protein kinase C to myogenic tone in rat cerebral arteries in hypertension.

Arterial smooth muscle constriction in response to pressure, i.e., myogenic tone, may involve calcium-dependent and calcium-sensitization mechanisms. Calcium sensitization in vascular smooth muscle is regulated by kinases such as PKC and Rho kinase, and activity of these kinases is known to be altered in cardiovascular disorders. In the present study, we evaluated the relative contribution of PKC and Rho kinase to myogenic tone in cerebral arteries in hypertension. Myogenic tone and arterial wall calcium in Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were measured simultaneously, and the effect of PKC and Rho kinase inhibitors on myogenic tone was evaluated. SHR arteries showed significantly greater myogenic tone than WKY arteries. Pressure/wall tension-arterial wall calcium curves showed a hyperbolic relation in WKY rats, but the curves for SHR arteries were parabolic. Myogenic tone was decreased by the Rho kinase inhibitors Y-27632 and HA-1077, with a significantly greater effect in SHR than in WKY arteries. Reduction in myogenic tone produced by the PKC inhibitor bisindolylmaleimide I in WKY and SHR arteries was significantly less than that produced by Rho kinase inhibition. The pressure-dependent increase in myogenic tone was significantly decreased by Y-27632, and the decrease was markedly greater than that produced by bisindolylmaleimide I in SHR arteries. In WKY arteries, the pressure-dependent increase in myogenic tone was decreased to a similar extent by Y-27632 and bisindolylmaleimide I. These results suggest greater myogenic tone with increased calcium sensitization in SHR arteries, largely because of Rho kinase activation, with a minor contribution of PKC activation.

Transforming growth factor-beta1 increases CXCR4 expression, stromal-derived factor-1alpha-stimulated signalling and human immunodeficiency virus-1 entry in human monocyte-derived macrophages.

Stromal-derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 play crucial roles in leukocyte migration and activation, as well as embryogenesis, angiogenesis, cancer and viral pathogenesis. CXCR4 is one of the major human immunodeficiency virus-1 (HIV-1) coreceptors on macrophages. In many tissues macrophages are one of the predominant cell types infected by HIV-1 and act as a reservoir for persistent infection and viral dissemination. In patients infected by HIV-1, blood and tissue levels of transforming growth factor-beta1 (TGF-beta1) are increased. The purpose of this study was to evaluate the effects of TGF-beta1 on CXCR4 expression and function in primary human monocyte-derived macrophages (MDMs) and rat microglia. TGF-beta1 up-regulated CXCR4 and enhanced SDF-1alpha-stimulated ERK1,2 phosphorylation in these cells. The increased CXCR4 expression in human MDMs resulted in increased susceptibility of the cells to entry by dual-tropic CXCR4-using HIV-1 (D-X4). In contrast, TGF-beta1 failed to increase CCR5 expression or infection by a CCR5-using virus in MDMs. Our data demonstrate that TGF-beta1 enhances macrophage responsiveness to SDF-1alpha stimulation and susceptibility to HIV-1 by selectively increasing expression of CXCR4. The results suggest that increased expression of CXCR4 on macrophages may contribute to the emergence of dual-tropic X4 viral variants at later stages of HIV-1 infection.

Twenty years of calcium imaging: cell physiology to dye for.

The use of fluorescent dyes over the past two decades has led to a revolution in our understanding of calcium signaling. Given the ubiquitous role of Ca(2+) in signal transduction at the most fundamental levels of molecular, cellular, and organismal biology, it has been challenging to understand how the specificity and versatility of Ca(2+) signaling is accomplished. In excitable cells, the coordination of changing Ca(2+) concentrations at global (cellular) and well-defined subcellular spaces through the course of membrane depolarization can now be conceptualized in the context of disease processes such as cardiac arrhythmogenesis. The spatial and temporal dimensions of Ca(2+) signaling are similarly important in non-excitable cells, such as endothelial and epithelial cells, to regulate multiple signaling pathways that participate in organ homeostasis as well as cellular organization and essential secretory processes.

Increased atrial and brain natriuretic peptides in adults with cyanotic congenital heart disease: enhanced understanding of the relationship between hypoxia and natriuretic peptide secretion.

BACKGROUND: Brain natriuretic peptide (BNP) levels are used in the evaluation of patients with heart disease, yet there is little understanding of the effect of hypoxia on natriuretic peptide secretion. Furthermore, recent data suggest that oxytocin may mediate stretch-induced atrial natriuretic peptide (ANP) secretion. METHODS AND RESULTS: Ten patients with cyanotic congenital heart defects and 10 control subjects were studied. N-terminal proatrial natriuretic peptide and N-terminal probrain natriuretic peptide levels were 4-fold (P=0.02) and 12-fold (P=0.03) greater in cyanotic patients than in control subjects. Cyanotic patients had reduced body water compared with control subjects, although the difference did not reach statistical significance (P=0.22). In a separate group of patients, cardiac myocytes were isolated from the right atrial appendage during CABG. The amount of oxygen in the buffered saline was varied to simulate hypoxia. Isolated hypoxic atrial myocytes had 43% fewer dense surface secretory granules compared with normoxic myocytes (P<0.0001). Immunohistochemical staining demonstrated decreased ANP and BNP in hypoxic compared with normoxic right atrial tissue. Isolated myocytes also degranulated when incubated with oxytocin (P<0.0001), but there was no difference in oxytocin levels in cyanotic patients compared with control subjects (P=0.49). CONCLUSIONS: ANP and BNP are markedly elevated in adults with cyanotic congenital heart disease despite reduced body water. Our results show that hypoxia is a direct stimulus for ANP and BNP secretion in human cardiac myocytes. These findings may have implications for the interpretation of BNP levels in the assessment of patients with heart and lung disease.

Prevention of cardiac hypertrophy by angiotensin II type-2 receptor gene transfer.

The role of the angiotensin II type-2 receptor (AT2R) in cardiac hypertrophy remains elusive despite its demonstrated involvement in cardiovascular development. We have previously shown that a lentiviral vector gene delivery system is able to transduce cardiac tissue with high efficiency in vivo. Using such an approach, our objectives in the present study were 2-fold: (1) to overexpress the AT2R in cardiac tissue after completion of natural embryonic development of the heart and (2) to determine the effects of this overexpression on cardiac hypertrophy and basal blood pressure (BP). A lentiviral vector encoding the AT2R (lenti-AT2R) was administered (1.5x10(8) transducing units) into the left ventricular space of 5-day-old spontaneously hypertensive rats (SHRs). AT2R transgene expression increased in these animals and persisted for 30 weeks. In contrast, the expression of the angiotensin II type-1 receptor remained unchanged following lenti-AT2R treatment. At 21 weeks following gene transduction, the lenti-AT2R-treated SHRs exhibited decreased left ventricular wall thickness compared with control animals. In contrast, basal BP did not differ between the two SHR groups. Finally, heart weight to body weight ratios indicated a significant decrease in lenti-AT2R-treated SHRs compared with SHR controls. Our data indicate that AT2R overexpression attenuates cardiac hypertrophy in the SHR. This beneficial outcome was observed despite the existence of elevated BP.

Quantification of the rat left ventricle force and Ca2+ -frequency relationships: similarities to dog and human.

OBJECTIVE: To measure and quantify the force-frequency (FFR) and Ca(2+)-frequency (CaFR) relationships in isolated rat left ventricular (LV) muscle at physiological heart rates and compare the obtained FFR to that measured in larger mammalian muscle from dog and human using the same experimental protocol. METHODS: Rat papillary muscle was isolated from the LV of adult male Sprague-Dawley rats, and dog and human muscles were from free-wall LV biopsies, loaded with the Ca(2+) indicator Fura-2, allowed to recover from isolation trauma and then subjected to direct electrical stimulation while measuring force production and intracellular Ca(2+) transients. RESULTS: We obtained a positive FFR between 1 and 4 Hz that is qualitatively similar to that found in isolated LV epicardial muscle strips from dogs and humans with normal LV function. The FFR reflects the cytosolic Ca(2+) transients in amplitude. Isoproterenol yielded an enhancement in force, but flattening of the FFR, whereas cyclopiazonic acid caused depression of FFR amplitude without changing frequency-dependent shape. CONCLUSION: We describe an experimental protocol that consistently yields positive FFRs in rat, dog and human LV muscle at stimulation rates between 1 and 4 Hz, without significant qualitative differences. We attribute previously observed negative FFR in rat muscle to an increase in SERCA activity early after excision and preparation of the muscle strips.

Myogenic tone and reactivity of the rat ophthalmic artery.

PURPOSE: To study and quantify myogenic behavior and reactivity of the rat ophthalmic artery to pressure and different vasoactive substances in vitro. METHODS: Rat ophthalmic arteries (diameter of 217 +/- 6 microm, n = 22) were isolated, cannulated with glass pipettes in an arteriograph and pressurized in a physiological buffer. Internal diameter was continuously monitored. The effect of intraluminal pressure on the diameter was assessed and concentration-response curves to different constrictor and dilator agonists were obtained at an intraluminal pressure of 70 mm Hg. RESULTS: Myogenic tone developed at an intraluminal pressure of 30 to 40 mm Hg, continued to increase, and was maintained up to a pressure of 199 mm Hg in these arteries. Arteries dilated and constricted in response to 16 and 60 mM potassium, respectively. Endothelin-1 was the most potent and efficacious constrictor, with a biphasic concentration-response curve, followed by vasopressin, serotonin, U-46619 and phenylephrine. Carbachol was the most efficacious dilator, followed by isoprenaline. The peptide dilators calcitonin gene-related peptide (CGRP) and vasoactive intestinal peptide (VIP) were potent but less efficacious than carbachol and isoprenaline. Histamine and adenosine were even less potent and less efficacious dilators. NG-nitro-L-arginine methyl ester (L-NAME) constricted and indomethacin dilated the arteries. CONCLUSIONS: This study provides the first direct evidence for myogenic autoregulatory properties and pharmacological heterogeneity in the rat ophthalmic artery.

Inhibitors of gap junctions attenuate myogenic tone in cerebral arteries.

The effects of two structurally distinct inhibitors of gap junction communication were studied by using three different forms of vasoconstriction in pressurized rat middle cerebral arteries. The sensitivity of myogenic tone (at 60 mmHg), vasopressin-induced tone (10 nM, at 20 mmHg), and depolarizing solution-induced tone (80 mM K(+), at 20 mmHg) to inhibition by heptanol (1.0 microM to 3.0 mM) or 18alpha-glycyrrhetinic acid (18alpha-GA, 1.0 to 50 microM) were determined. Pressure-induced myogenic tone was inhibited by heptanol (IC(50) = 0.75 +/- 0.09 mM) and 18alpha-GA ( approximately 30 microM). Vasopressin-induced vasoconstriction was also inhibited by heptanol (IC(50) = 0.4 +/- 0.3 mM) and 18alpha-GA (>1 microM). Depolarizing solution-induced vasoconstriction was less sensitive to inhibition by heptanol compared to vasopressin (P < 0.01) or pressure-induced constriction (P < 0.05). However, 18alpha-GA did not inhibit depolarization-induced constriction. Sharp microelectrode experiments on isolated arteries revealed stable membrane potentials, with no detectable effect of heptanol (1 mM) or 18alpha-GA (20-30 microM) on the average membrane potential at 20 mmHg. However, approximately 20% of impaled cells (5 of 28) exhibited uncharacteristic oscillations in membrane potential after pharmacological uncoupling. At 60 mmHg a approximately 7- to 9-mV hyperpolarization and corresponding vasodilation (approximately 50%) was observed, and the frequency of membrane potential oscillations doubled (9 of 23 cells). These data indicate that gap junctions play an important role in the maintenance and modulation of membrane potential and tone in cerebral resistance arteries.

Role of phospholipase C in development of myogenic tone in rat posterior cerebral arteries.

Earlier studies have implicated phospholipase C (PLC) in the development of myogenic tone (MT) based on pharmacological studies in larger arteries. In the present study, we further investigated the cellular effects of PLC inhibition using pharmacological and electrophysiological approaches to provide more quantitative functional evidence for the involvement of PLC in the genesis of MT in small cerebral arteries. The phosphatidylinositol-selective PLC (PI-PLC) inhibitor U-73122 decreased MT by 87% in posterior cerebral arteries from Sprague-Dawley rats with pIC(50) of 6.2 +/- 0.09 (n = 5). Similar potency (pIC(50) of 6.2 +/- 0.04, n = 5) was observed in arteries with MT that were further constricted with 30 nM serotonin. The phosphatidylcholine-specific (PC-PLC) inhibitor D609 had no effect on MT. U-73343, the inactive analog of U-73122, did not show any relaxant effect, but at higher concentrations (>1 microM) it reduced MT. In the presence of 125-500 nM U-73122, the pressure-diameter curves shifted toward that obtained in Ca-free conditions. U-73122-mediated decrease in MT was accompanied by a decrease in mean arterial wall calcium (maximum effect: 77 +/- 3% of 16 mM KCl-mediated decrease, n = 4). This was due to a simultaneous membrane potential hyperpolarization of approximately 9 mV or from -44 +/- 1 to -53 +/- 2 mV (10 microM, P < 0.001, n = 8). In summary, this study provides the first quantitative data suggesting a critical importance of PI-PLC in the genesis of pressure-induced MT in rat cerebral arteries via membrane potential depolarization and increased calcium influx.