Nathan R. Tykocki

The interdependence of endothelin-1 and calcium: a review

The 21-amino-acid peptide ET-1 (endothelin-1) regulates a diverse array of physiological processes, including vasoconstriction, angiogenesis, nociception and cell proliferation. Most of the effects of ET-1 are associated with an increase in intracellular calcium concentration. The calcium influx and mobilization pathways activated by ET-1, however, vary immensely. The present review begins with the basics of calcium signalling and investigates the different ways intracellular calcium concentration can increase in response to a stimulus. The focus then shifts to ET-1, and discusses how ET receptors mobilize calcium. We also examine how disease alters calcium-dependent responses to ET-1 by discussing changes to ET-1-mediated calcium signalling in hypertension, as there is significant interest in the role of ET-1 in this important disease. A list of unanswered questions regarding ET-mediated calcium signals are also presented, as well as perspectives for future research of calcium mobilization by ET-1.

Endothelin ETB Receptors in Arteries and Veins: Multiple Actions in the Vein

Endothelin receptors (ETA and ETB) mediate responses to ET-1. ETB receptor function seems to differ between a similarly sized arterial and venous pair, the rat vena cava (RVC) and rat thoracic aorta (RA). ETB receptors mediate RVC contraction directly, but it is unclear whether ETB receptors mediate contraction in RA. Because of these apparent differences in ETB receptor-mediated vascular contraction, we hypothesize that relaxant ETB-receptor mechanisms in RVC would be different from those in RA. RA and RVC rings were isolated from rats for measurement of isometric contraction. When contracted with prostaglandin F-2α (PGF-2α) (20 μM), the ETB receptor agonist sarafotoxin-6c (S6c) (100 nM) significantly relaxed RA and RVC. Nω-Nitro-l-arginine (LNNA) (100 μM) or endothelial denudation abolished relaxation to S6c in RA. By contrast, S6c-induced relaxation of RVC was attenuated but not abolished by LNNA and endothelial denudation. RVC (PGF-2α-contracted) relaxed to low concentrations of ET-1, whereas under the same conditions RA responded with contraction. ET-1-induced relaxation in RA was observed only with ETA receptor blockade. Vessels from dopamine-β-hydroxylase-ETB transgenic rats, which lack functional ETB receptors in the vasculature, were also used. RVC (PGF-2α-contracted) from these rats did not relax to ET-1. Thus, although both RA and RVC possess endothelial relaxant ETB receptors, RA and RVC differ in that relaxant ETB receptors may also be present in smooth muscle of RVC. Moreover, the mechanisms of endothelial cell ETB receptor-mediated relaxation in RA and RVC are not the same.

Smooth Muscle Ion Channels and Regulation of Vascular Tone in Resistance Arteries and Arterioles.

Vascular tone of resistance arteries and arterioles determines peripheral vascular resistance, contributing to the regulation of blood pressure and blood flow to, and within the bodys tissues and organs. Ion channels in the plasma membrane and endoplasmic reticulum of vascular smooth muscle cells (SMCs) in these blood vessels importantly contribute to the regulation of intracellular Ca2+ concentration, the primary determinant of SMC contractile activity and vascular tone. Ion channels provide the main source of activator Ca2+ that determines vascular tone, and strongly contribute to setting and regulating membrane potential, which, in turn, regulates the open-state-probability of voltage gated Ca2+ channels (VGCCs), the primary source of Ca2+ in resistance artery and arteriolar SMCs. Ion channel function is also modulated by vasoconstrictors and vasodilators, contributing to all aspects of the regulation of vascular tone. This review will focus on the physiology of VGCCs, voltage-gated K+ (KV) channels, large-conductance Ca2+-activated K+ (BKCa) channels, strong-inward-rectifier K+ (KIR) channels, ATP-sensitive K+ (KATP) channels, ryanodine receptors (RyRs), inositol 1,4,5-trisphosphate receptors (IP3Rs), and a variety of transient receptor potential (TRP) channels that contribute to pressure-induced myogenic tone in resistance arteries and arterioles, the modulation of the function of these ion channels by vasoconstrictors and vasodilators, their role in the functional regulation of tissue blood flow and their dysfunction in diseases such as hypertension, obesity, and diabetes.

Transient contractions of urinary bladder smooth muscle are drivers of afferent nerve activity during filling

As the urinary bladder fills, the rate of rise (but not the frequency) of spontaneous contractions of smooth muscle in the bladder wall increases, thereby eliciting increased afferent nerve activity that signals the fullness of the bladder.

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum – if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

One-month serotonin infusion results in a prolonged fall in blood pressure in the deoxycorticosterone acetate (DOCA) salt hypertensive rat.

A 7-day infusion of serotonin (5-hydroxytryptamine, 5-HT) causes a sustained fall in elevated blood pressure in the male deoxycorticosterone acetate (DOCA)-salt rat. As hypertension is a long-term disease, we presently test the hypothesis that a longer (30 day) 5-HT infusion could cause a sustained fall in blood pressure in the established hypertensive DOCA-salt rat. This time period (∼4 weeks) was also sufficient to test whether 5-HT could attenuate the development of DOCA-salt hypertension. 5-HT (25 μg/kg/min; sc) or vehicle (Veh) was delivered via osmotic pump to (1) established DOCA-salt rats for one month, (2) Sprague-Dawley rats prior to DOCA-salt administration for one month, and blood pressure and heart rate measured telemetrically. On the final day of 5-HT infusion, free platelet poor plasma 5-HT concentrations were significantly higher in 5-HT versus Veh-infused rats, and mean arterial pressure was significantly lower in 5-HT-infused (135 ± 4 mmHg vs Veh-infused 151 ± 7 mmHg) established DOCA-salt rats. By contrast, 5-HT-infusion did not prevent the development of DOCA-salt hypertension (144 ± 7 mmHg vs Veh = 156 ± 6 mmHg). Isometric contraction of aortic strips was measured, and neither the potency nor maximum contraction to the alpha adrenergic receptor agonist phenylephrine (PE) or 5-HT were modified by infusion of 5-HT (established or preventative infusion), and maximum aortic relaxation to acetylcholine (ACh) was modestly but not significantly enhanced (∼15% improvement). This study demonstrates 5-HT is capable of lowering blood pressure in established DOCA-salt hypertensive rats over the course of one month in a mechanism that does not significantly modify or is dependent on modified vascular responsiveness. This finding opens the possibility that elevation of 5-HT levels could be useful in the treatment of hypertension.

Divergent signaling mechanisms for venous versus arterial contraction as revealed by endothelin-1

OBJECTIVE Venous function is underappreciated in its role in blood pressure determination, a physiologic parameter normally ascribed to changes in arterial function. Significant evidence points to the hormone endothelin-1 (ET-1) as being important to venous contributions to blood pressure. We hypothesized that the artery and vein should similarly depend on the signaling pathways stimulated by ET-1, specifically phospholipase C (PLC) activation. This produces two functional arms of signaling: diacylglycerol (DAG; protein kinase C [PKC] activation) and inositol trisphosphate (IP3) production (intracellular calcium release). METHODS The model was the male Sprague-Dawley rat. Isolated tissue baths were used to measure isometric contraction. Western blot and immunocytochemical analyses measured the magnitude of expression and site of expression, respectively, of IP3 receptors in smooth muscle/tissue. Pharmacologic methods were used to modify PLC activity and signaling elements downstream of PLC (IP3 receptors, PKC). RESULTS ET-1-induced contraction was PLC dependent in both tissues as the PLC inhibitor U-73122 significantly reduced contraction in aorta (86% ± 4% of control; P < .05) and vena cava (49% ± 11% of control; P < .05). However, ET-1-induced contraction was not significantly inhibited by the IP3 receptor inhibitor 2-aminoethoxydiphenylborane (100 μM) in vena cava (82% ± 8% of control; P = .23) but was in the aorta (55% ± 4% of control; P < .05). All three IP3 receptor isoforms were located in venous smooth muscle. IP3 receptors were functional in both tissues as the novel membrane-permeable IP3 analogue (Bt-IP3; 10 μM) contracted aorta and vena cava. Similarly, whereas the PKC inhibitor chelerythrine (10 μM) attenuated ET-1-induced contraction in vena cava and aorta (5% ± 2% and 50% ± 5% of control, respectively; P < .05), only the vena cava contracted to the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol. CONCLUSIONS These findings suggest that ET-1 activates PLC in aorta and vena cava, but vena cava contraction to ET-1 may be largely IP3 independent. Rather, DAG—not IP3—may contribute to contraction to ET-1 in vena cava, in part by activation of PKC. These studies outline a fundamental difference between venous and arterial smooth muscle and further reinforce a heterogeneity of vascular smooth muscle function that could be taken advantage of for therapeutic development.

Social stress in mice induces urinary bladder overactivity and increases TRPV1 channel-dependent afferent nerve activity

Social stress has been implicated as a cause of urinary bladder hypertrophy and dysfunction in humans. Using a murine model of social stress, we and others have shown that social stress leads to bladder overactivity. Here, we show that social stress leads to bladder overactivity, increased bladder compliance, and increased afferent nerve activity. In the social stress paradigm, 6-wk-old male C57BL/6 mice were exposed for a total of 2 wk, via barrier cage, to a C57BL/6 retired breeder aggressor mouse. We performed conscious cystometry with and without intravesical infusion of the TRPV1 inhibitor capsazepine, and measured pressure-volume relationships and afferent nerve activity during bladder filling using an ex vivo bladder model. Stress leads to a decrease in intermicturition interval and void volume in vivo, which was restored by capsazepine. Ex vivo studies demonstrated that at low pressures, bladder compliance and afferent activity were elevated in stressed bladders compared with unstressed bladders. Capsazepine did not significantly change afferent activity in unstressed mice, but significantly decreased afferent activity at all pressures in stressed bladders. Immunohistochemistry revealed that TRPV1 colocalizes with CGRP to stain nerve fibers in unstressed bladders. Colocalization significantly increased along the same nerve fibers in the stressed bladders. Our results support the concept that social stress induces TRPV1-dependent afferent nerve activity, ultimately leading to the development of overactive bladder symptoms.

Reverse-mode Na+/Ca2+ exchange is an important mediator of venous contraction.

The Na(+)/Ca(2+) exchanger (NCX) is a bi-directional regulator of cytosolic Ca(2+), causing Ca(2+) efflux in forward-mode and Ca(2+) influx in reverse-mode. We hypothesized that reverse-mode NCX is a means of Ca(2+) entry in rat aorta (RA) and vena cava (RVC). NCX protein in RA and RVC was confirmed by immunoprecipitation. To assess NCX function, isometric contraction and intracellular Ca(2+) was measured in RA and RVC rings in response to low extracellular Na(+), endothelin-1 (ET-1), and KCl, in the presence or absence of the NCX antagonist KB-R7943. In RVC, low extracellular Na(+) caused vasoconstriction and an increase in intracellular Ca(2+) that was attenuated by 10μM KB-R7943. KB-R7943 (10 μM) attenuated maximal contraction to ET-1 in RVC (53 ± 9% of control), but not RA (91±1% of control). KB-R7943 (10 μM) reduced the maximal contraction to KCl in RA (48 ± 5%) and nearly abolished it in RVC (9 ± 2%), suggesting that voltage-dependent Ca(2+) influx may be inhibited by KB-R7943 as well. However, the L-type Ca(2+) channel inhibitor nifedipine (1 μM) did not alter ET-1-induced contraction. Our findings suggest that reverse-mode NCX is an important mechanism of Ca(2+) influx in RVC but not RA, especially during ET-1-induced contraction. Also, the effects of KB-R7943 on ET-1-induced contraction of RA and RVC are predominantly mediated by reverse-mode NCX inhibition and not due to off-target inhibition of Ca(2+) channels.

Location, Location, Location: Juxtaposed calcium-signaling microdomains as a novel model of the vascular smooth muscle myogenic response

Small arteries and arterioles constrict in response to increases in intraluminal pressure through a process called “myogenic tone” or the “myogenic response” ([McCarron et al., 1989][1]). Development of myogenic tone has profound physiological implications; it contributes substantially to