A mathematical model of the myogenic response to systolic pressure in the afferent arteriole

Elevations in systolic blood pressure are believed to be closely linked to the pathogenesis and progression of renal diseases. It has been hypothesized that the afferent arteriole (AA) protects the glomerulus from the damaging effects of hypertension by sensing increases in systolic blood pressure and responding with a compensatory vasoconstriction (Loutzenhiser R, Bidani A, Chilton L. Circ Res 90: 1316-1324, 2002). To investigate this hypothesis, we developed a mathematical model of the myogenic response of an AA wall, based on an arteriole model (Gonzalez-Fernandez JM, Ermentrout B. Math Biosci 119: 127-167, 1994). The model incorporates ionic transport, cell membrane potential, contraction of the AA smooth muscle cell, and the mechanics of a thick-walled cylinder. The model represents a myogenic response based on a pressure-induced shift in the voltage dependence of calcium channel openings: with increasing transmural pressure, model vessel diameter decreases; and with decreasing pressure, vessel diameter increases. Furthermore, the model myogenic mechanism includes a rate-sensitive component that yields constriction and dilation kinetics similar to behaviors observed in vitro. A parameter set is identified based on physical dimensions of an AA in a rat kidney. Model results suggest that the interaction of Ca(2+) and K(+) fluxes mediated by voltage-gated and voltage-calcium-gated channels, respectively, gives rise to periodicity in the transport of the two ions. This results in a time-periodic cytoplasmic calcium concentration, myosin light chain phosphorylation, and cross-bridge formation with the attending muscle stress. Furthermore, the model predicts myogenic responses that agree with experimental observations, most notably those which demonstrate that the renal AA constricts in response to increases in both steady and systolic blood pressures. The myogenic model captures these essential functions of the renal AA, and it may prove useful as a fundamental component in a multiscale model of the renal microvasculature suitable for investigations of the pathogenesis of hypertensive renal diseases.     

Role of vascular potassium channels in the regulation of renal hemodynamics

K(+) conductance is a major determinant of membrane potential (V(m)) in vascular smooth muscle (VSMC) and endothelial cells (EC). The vascular tone is controlled by V(m) through the action of voltage-operated Ca(2+) channels (VOCC) in VSMC. Increased K(+) conductance leads to hyperpolarization and vasodilation, while inactivation of K(+) channels causes depolarization and vasoconstriction. K(+) channels in EC indirectly participate in the control of vascular tone by several mechanisms, e.g., release of nitric oxide and endothelium-derived hyperpolarizing factor. In the kidney, a change in the activity of one or more classes of K(+) channels will lead to a change in hemodynamic resistance and therefore of renal blood flow and glomerular filtration pressure. Through these effects, the activity of renal vascular K(+) channels influences renal salt and water excretion, fluid homeostasis, and ultimately blood pressure. Four main classes of K(+) channels [calcium activated (K(Ca)), inward rectifier (K(ir)), voltage activated (K(V)), and ATP sensitive (K(ATP))] are found in the renal vasculature. Several in vitro experiments have suggested a role for individual classes of K(+) channels in the regulation of renal vascular function. Results from in vivo experiments are sparse. We discuss the role of the different classes of renal vascular K(+) channels and their possible role in the integrated function of the renal microvasculature. Since several pathological conditions, among them hypertension, are associated with alterations in K(+) channel function, the role of renal vascular K(+) channels in the control of salt and water excretion deserves attention.     

Diacylglycerol and protein kinase C activate cation channels involved in myogenic tone

The smooth muscle cells of resistance arteries depolarize and contract when intravascular pressure is elevated. This is a central characteristic of myogenic tone, which plays an important role in regulation of blood flow in many vascular beds. Pressure-induced vascular smooth muscle depolarization depends in part on the activation of cation channels. Here, we show that activation of these smooth muscle cation channels and pressure-induced depolarization are mediated by protein kinase C in cerebral resistance arteries. Diacylglycerol, phorbol myristate acetate, and cell swelling activate a cation current that we have previously shown is mediated by transient receptor potential channels. These currents, as well as the smooth muscle cell depolarizations of intact arteries induced by diacylglycerol, phorbol ester, and elevation of intravascular pressure, are nearly eliminated by protein kinase C inhibitors. These results suggest a major mechanism of myogenic tone involves mechanotransduction through phospholipase C, diacylglycerol production, and protein kinase C activation, which increase cation channel activity. The associated depolarization activates L-type calcium channels, leading to increased intracellular calcium and vasoconstriction.     

Control and Modulation of Fluid Flow in the Rat Kidney

We have developed a mathematical model of the rat’s renal hemodynamics in the nephron level, and used that model to study flow control and signal transduction in the rat kidney. The model represents an afferent arteriole, glomerular filtration, and a segment of a short-loop nephron. The model afferent arteriole is myogenically active and represents smooth muscle membrane potential and electrical coupling. The myogenic mechanism is based on the assumption that the activity of nonselective cation channels is shifted by changes in transmural pressure, such that elevation in pressure induces vasoconstriction, which increases resistance to blood flow. From the afferent arteriole’s fluid delivery output, glomerular filtration rate is computed, based on conservation of plasma and plasma protein. Chloride concentration is then computed along the renal tubule based on solute conservation that represents water reabsorption along the proximal tubule and the water-permeable segment of the descending limb, and chloride fluxes driven by passive diffusion and active transport. The model’s autoregulatory response is predicted to maintain stable renal blood flow within a physiologic range of blood pressure values. Power spectra associated with time series predicted by the model reveal a prominent fundamental peak at ～165 mHz arising from the afferent arteriole’s spontaneous vasomotion. Periodic external forcings interact with vasomotion to introduce heterodynes into the power spectra, significantly increasing their complexity.     

Modified cardiovascular L-type channels in mice lacking the voltage-dependent Ca2+ channel beta3 subunit

The beta subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming alpha1 subunits. Of the four beta subunits reported to date, the beta3 subunit is highly expressed in smooth muscle cells and is thought to consist of L-type calcium channels. To determine the role of the beta3 subunit in the voltage-dependent calcium channels of the cardiovascular system in situ, we performed a series of experiments in beta3-null mice. Western blot analysis indicated a significant reduction in expression of the alpha1 subunit in the plasma membrane of beta3-null mice. Dihydropyridine binding experiments also revealed a significant decrease in the calcium channel population in the aorta. Electrophysiological analyses indicated a 30% reduction in Ca2+ channel current density, a slower inactivation rate, and a decreased dihydropyridine-sensitive current in beta3-null mice. The reductions in the peak current density and inactivation rate were reproduced in vitro by co-expression of the calcium channel subunits in Chinese hamster ovary cells. Despite the reduced channel population, beta3-null mice showed normal blood pressure, whereas a significant reduction in dihydropyridine responsiveness was observed. A high salt diet significantly elevated blood pressure only in the beta3-null mice and resulted in hypertrophic changes in the aortic smooth muscle layer and cardiac enlargement. In conclusion, this study demonstrates the involvement and importance of the beta3 subunit of voltage-dependent calcium channels in the cardiovascular system and in regulating channel populations and channel properties in vascular smooth muscle cells.     

Quick and effective hyperpolarization of the membrane potential in intact smooth muscle cells of blood vessels by synchronization modulation electric field

Blood vessel dilation starts from activation of the Na/K pumps and inward rectifier K channels in the vessel smooth muscle cells, which hyperpolarizes the cell membrane potential and closes the Ca channels. As a result, the intracellular Ca concentration reduces, and the smooth muscle cells relax and the blood vessel dilates. Activation of the Na/K pumps and the membrane potential hyperpolarization plays a critical role in blood vessel functions. Previously, we developed a new technique, synchronization modulation, to control the pump functions by electrically entraining the pump molecules. We have applied the synchronization modulation electric field noninvasively to various intact cells and demonstrated the field-induced membrane potential hyperpolarization. We further applied the electric field to blood vessels and investigated the field induced functional changes of the vessels. In this paper, we report the results in a study of the membrane potential change in the smooth muscle cells of mesenteric blood vessels in response to the oscillating electric field. We found that the synchronization modulation electric field can effectively hyperpolarize the muscle membrane potential quickly in seconds under physiological conditions.     

Effects of arterial wall stress on vasomotion

Smooth muscle and endothelial cells in the arterial wall are exposed to mechanical stress. Indeed blood flow induces intraluminal pressure variations and shear stress. An increase in pressure may induce a vessel contraction, a phenomenon known as the myogenic response. Many muscular vessels present vasomotion, i.e., rhythmic diameter oscillations caused by synchronous cytosolic calcium oscillations of the smooth muscle cells. Vasomotion has been shown to be modulated by pressure changes. To get a better understanding of the effect of stress and in particular pressure on vasomotion, we propose a model of a blood vessel describing the calcium dynamics in a coupled population of smooth muscle cells and endothelial cells and the consequent vessel diameter variations. We show that a rise in pressure increases the calcium concentration. This may either induce or abolish vasomotion, or increase its frequency depending on the initial conditions. In our model the myogenic response is less pronounced for large arteries than for small arteries and occurs at higher values of pressure if the wall thickness is increased. Our results are in agreement with experimental observations concerning a broad range of vessels.     

E3-targeted anti-TRPC5 antibody inhibits store-operated calcium entry in freshly isolated pial arterioles

Smooth muscle cells in arterioles have pivotal roles in the determination of blood pressure and distribution of local blood flow. The cells exhibit calcium entry in response to passive store depletion, but the mechanisms and relevance of this phenomenon are poorly understood. Previously, a role for canonical transient receptor potential 1 (TRPC1) was indicated, but heterologous expression studies showed TRPC1 to have poor function in isolation, suggesting a requirement for additional proteins. Here we test the hypothesis that TRPC5 is such an additional protein, because TRPC5 forms heteromultimeric channels with TRPC1, and RNA encoding TRPC5 is present in arterioles. Recordings were from arteriolar fragments freshly isolated from rabbit pial membrane. Ionic current in response to store depletion has properties like that of the TRPC1/TRPC5 heteromultimer, and so the effect of the E3-targeted, externally acting, anti-TRPC5 blocking antibody (T5E3) was explored. T5E3 suppressed calcium entry in store-depleted arterioles but had no effect in the absence of store depletion. T5E3 preadsorbed to its antigenic peptide did not inhibit calcium entry. TRPC6 is commonly detected in smooth muscle and is present in the arterioles, but T5E3 had no effect on TRPC6. The data suggest that calcium entry occurring in response to passive store depletion in smooth muscle cells of arterioles involves TRPC5 as well as TRPC1.     

Mechanisms of Propagation of Intercellular Calcium Waves in Arterial Smooth Muscle Cells

In rat mesenteric arteries, smooth muscle cells exhibit intercellular calcium waves in response to local phenylephrine stimulation. These waves have a velocity of ∼20 cells/s and a range of ∼80 cells. We analyze these waves in a theoretical model of a population of coupled smooth muscle cells, based on the hypothesis that the wave results from cell membrane depolarization propagation. We study the underlying mechanisms and highlight the importance of voltage-operated channels, calcium-induced calcium release, and chloride channels. Our model is in agreement with experimental observations, and we demonstrate that calcium waves presenting a velocity of ∼20 cells/s can be mediated by electrical coupling. The wave velocity is limited by the time needed for calcium influx through voltage-operated calcium channels and the subsequent calcium-induced calcium release, and not by the speed of the depolarization spreading. The waves are partially regenerated, but have a spatial limit in propagation. Moreover, the model predicts that a refractory period of calcium signaling may significantly affect the wave appearance.     

Stimulation of arterial smooth muscle L-type calcium channels by hydrogen peroxide requires protein kinase C

Changes in intracellular calcium regulate countless biological processes. In arterial smooth muscle, voltage-dependent L-type calcium channels are major conduits for calcium entry with the primary function being determination of arterial diameter. Similarly, changes in intracellular redox status, either discrete controlled changes or global pathological perturbations, are also critical determinants of cell function. We recently reported that in arterial smooth muscle cells, local generation of hydrogen peroxide leads to colocalized calcium entry through L-type calcium channels. Here we extend our investigation into mechanisms linking hydrogen peroxide to calcium influx through L-type calcium channels by focusing on the role of protein kinase C (PKC). Our data indicate that stimulation of L-type calcium channels by hydrogen peroxide requires oxidant-dependent increases in PKC catalytic activity. This effect is independent of classical cofactor-dependent activation of PKC by diacylglycerol. These data provide additional experimental evidence supporting the concept of oxidative stimulation of L-type calcium channels.     

Calcium channels in smooth muscle cells

In smooth muscle cells, the electrophysiological properties of potential-dependent calcium channels are similar to those described in other excitable cells. The calcium current is dependent on the extracellular calcium concentration; it is insensitive to external sodium removal and tetrodotoxin application. Other ions (Ba2+, Sr2+, Na+) can flow through the calcium channel. This channel is blocked by Mn2+, Co2+, Cd2+ and by organic inhibitors. The inactivation mechanism is mediated by both the membrane potential and the calcium influx. Ca2+ ions can also penetrate into the cell through receptor-operated channels. These channels show a low ionic selectivity and are generally less sensitive to organic Ca-blockers than the potential-dependent calcium channels. The finding of specific channel inhibitors as well as the study of the biochemical pathways between receptor activation and channel opening are prerequisites to further characterization of receptor-operated channels.     

When S1P meets ATP

Changes in intracellular calcium regulate countless biological processes. In arterial smooth muscle, voltage-dependent L-type calcium channels are major conduits for calcium entry with the primary function being determination of arterial diameter. Similarly, changes in intracellular redox status, either discrete controlled changes or global pathological perturbations, are also critical determinants of cell function. We recently reported that in arterial smooth muscle cells, local generation of hydrogen peroxide leads to colocalized calcium entry through L-type calcium channels. Here we extend our investigation into mechanisms linking hydrogen peroxide to calcium influx through L-type calcium channels by focusing on the role of protein kinase C (PKC). Our data indicate that stimulation of L-type calcium channels by hydrogen peroxide requires oxidant-dependent increases in PKC catalytic activity. This effect is independent of classical cofactor-dependent activation of PKC by diacylglycerol. These data provide additional experimental evidence supporting the concept of oxidative stimulation of L-type calcium channels.     

Voltage-independent calcium influx in smooth muscle

In smooth muscle cells, agonists such as neurotransmitters or hormones can induce an increase in [Ca(2+)](i) via a release of intracellular stored calcium or/and an influx of extracellular calcium. The calcium entry pathway operates through a variety of plasmalemmal calcium channels which involve voltage-dependent and voltage-independent calcium channels. Voltage-independent calcium channels include (1) receptor-operated channels (ROCs) activated by agonist-receptor interaction and, in the majority of cases, the downstream signal transduction proteins, (2) store-operated channels (SOCs) activated by the emptying of intracellular Ca(2+) store (mainly the sarcoplasmic reticulum), (3) mechanosensitive or stretch-activated channels (SACs) activated by membrane stretch. Generally, voltage-independent calcium channels are calcium permeable non-selective cation channels with electrophysiological differences, complex regulatory mechanisms and pharmacology. Although the molecular identity of voltage-independent calcium channels is not yet fully elucidated, there are growing evidences that these channels correspond to a new family of membrane proteins encoded by mammalian homologues of specific transient receptor potential (TRP) genes. Several types of TRP proteins are ubiquitously expressed in smooth muscle cells and variations in the expression depend on tissue and species. More recently, other proteins such as Orai1 and STIM1 proteins have been also proposed as participating in the molecular identity of voltage-independent calcium channels. These channels control phenomena such as smooth muscle cells proliferation and/or contraction.     

Mechanical control of cation channels in the myogenic response

Microcirculatory vessel response to changes in pressure, known as the myogenic response, is a key component of a tissue's ability to regulate blood flow. Experimental studies have not clearly elucidated the mechanical signal in the vessel wall governing steady-state reduction in vessel diameter upon an increase in intraluminal pressure. In this study, a multiscale computational model is constructed from established models of vessel wall mechanics, vascular smooth muscle (VSM) force generation, and VSM Ca(2+) handling and electrophysiology to compare the plausibility of vessel wall stress or strain as an effective mechanical signal controlling steady-state vascular contraction in the myogenic response. It is shown that, at the scale of a resistance vessel, wall stress, and not stretch (strain), is the likely physiological signal controlling the steady-state myogenic response. The model is then used to test nine candidate VSM stress-controlled channel variants by fitting two separate sets of steady-state myogenic response data. The channel variants include nonselective cation (NSC), supplementary Ca(2+) and Na(+), L-type Ca(2+), and large conductance Ca(2+)-activated K(+) channels. The nine variants are tested in turn, and model fits suggest that stress control of Ca(2+) or Na(+) influx through NSC, supplementary Ca(2+) or Na(+), or L-type Ca(2+) channels is sufficient to produce observed steady-state diameter changes with pressure. However, simulations of steady-state VSM membrane potential, cytosolic Ca(2+), and Na(+) with pressure show only that Na(+) influx through NSC channel also generates known trends with increasing pressure, indicating that stress-controlled Na(+) influx through NSC is sufficient to generate the myogenic response.     

T-type Ca2+ channels in vascular smooth muscle: multiple functions

Vascular smooth muscle is a major constituent of the blood vessel wall, and its many functions depend on type and location of the vessel, developmental or pathological state, and environmental and chemical factors. Vascular smooth muscle cells (VSMCs) use calcium as a signal molecule for multiple functions. An important component of calcium signaling pathways is the entry of extracellular calcium via voltage-gated Ca2+ channels, which in vascular smooth muscle cells (VSMCs) are of two main types, the high voltage-activated (HVA) L-type and low voltage-activated (LVA) T-type channels. Whereas L-type channels function primarily to regulate Ca2+ entry for contraction, it is generally accepted that T-type Ca2+ channels do not contribute significantly to arterial vasoconstriction, with the possible exception of the renal microcirculation. T-type Ca2+ channels are also present in some veins that display spontaneous contractile activity, where they likely generate pacemaker activity. T-type Ca2+ channel expression has also been associated with normal and pathological proliferation of VSMCs, often stimulated by external cues in response to insult or injury. Expression of T-type channels has been linked to the G1 and S phases of the cell cycle, a period important for the signaling of gene expression necessary for cell growth, progression of the cell cycle and ultimately cell division. To better understand T-type Ca2+ channel functions in VSM, it will be necessary to develop new approaches that are specifically targeted to this class of Ca2+ channels and its individual members.     

Smooth muscle NOS, colocalized with caveolin-1, modulates contraction in mouse small intestine

Neuronal nitric oxide synthase (nNOS) in myenteric neurons is activated during peristalsis to produce nitric oxide which relaxes intestinal smooth muscle. A putative nNOS is also found in the membrane of intestinal smooth muscle cells in mouse and dog. In this study we studied the possible functions of this nNOS expressed in mouse small intestinal smooth muscle colocalized with caveolin-1(Cav-1). Cav-1 knockout mice lacked nNOS in smooth muscle and provided control tissues. 60 mM KCl was used to increase intracellular [Ca(2+)] through L-type Ca(2+) channel opening and stimulate smooth muscle NOS activity in intestinal tissue segments. An additional contractile response to LNNA (100 muM, NOS inhibitor) was observed in KCl-contracted tissues from control mice and was almost absent in tissues from Cav-1 knockout mice. Disruption of caveolae with 40 mM methyl-beta cyclodextrin in tissues from control mice led to the loss of Cav-1 and nNOS immunoreactivity from smooth muscle as shown by immunohistochemistry and a reduction in the response of these tissues to N-omega-nitro-L-arginine (LNNA). Reconstitution of membrane cholesterol using water soluble cholesterol in the depleted segments restored the immunoreactivity and the response to LNNA added after KCl. Nicardipine (1 muM) blocked the responses to KCl and LNNA confirming the role of L-type Ca(2+) channels. ODQ (1 muM, soluble guanylate cyclase inhibitor) had the same effect as inhibition of NOS following KCl. We conclude that the activation of nNOS, localized in smooth muscle caveolae, by calcium entering through L-type calcium channels triggers nitric oxide production which modulates muscle contraction by a cGMP-dependent mechanism.     

SNAP-25 inhibits L-type Ca2+ channels in feline esophagus smooth muscle cells

We recently reported that non-secretory gastrointestinal smooth muscle cells also possessed SNARE proteins, of which SNAP-25 regulated Ca(2+)-activated (K(Ca)) and delayed rectifier K(+) channels (K(V)). Voltage-gated, long lasting (L-type) calcium channels (L(Ca)) play an important role in excitation-contraction coupling of smooth muscle. Here, we show that SNAP-25 could also directly inhibit the L-type Ca(2+) channels in feline esophageal smooth muscle cells at the SNARE complex binding synprint site. SNARE proteins could therefore regulate additional cell actions other than membrane fusion and secretion, in particular, coordinated muscle membrane excitability and contraction, through their actions on membrane Ca(2+) and K(+) channels.     

Effects of hypoxia, anoxia, and metabolic inhibitors on KATP channels in rat femoral artery myocytes

Vascular ATP-sensitive potassium (KATP) channels have an important role in hypoxic vasodilation. Because KATP channel activity depends on intracellular nucleotide concentration, one hypothesis is that hypoxia activates channels by reducing cellular ATP production. However, this has not been rigorously tested. In this study we measured KATP current in response to hypoxia and modulators of cellular metabolism in single smooth muscle cells from the rat femoral artery by using the whole cell patch-clamp technique. KATP current was not activated by exposure of cells to hypoxic solutions (Po2 approximately 35 mmHg). In contrast, voltage-dependent calcium current and the depolarization-induced rise in intracellular calcium concentration ([Ca2+]i) was inhibited by hypoxia. Blocking mitochondrial ATP production by using the ATP synthase inhibitor oligomycin B (3 microM) did not activate current. Blocking glycolytic ATP production by using 2-deoxy-D-glucose (5 mM) also did not activate current. The protonophore carbonyl cyanide m-chlorophenylhydrazone (1 microM) depolarized the mitochondrial membrane potential and activated KATP current. This activation was reversed by oligomycin B, suggesting it occurred as a consequence of mitochondrial ATP consumption by ATP synthase working in reverse mode. Finally, anoxia induced by dithionite (0.5 mM) also depolarized the mitochondrial membrane potential and activated KATP current. Our data show that: 1) anoxia but not hypoxia activates KATP current in femoral artery myocytes; and 2) inhibition of cellular energy production is insufficient to activate KATP current and that energy consumption is required for current activation. These results suggest that vascular KATP channels are not activated during hypoxia via changes in cell metabolism. Furthermore, part of the relaxant effect of hypoxia on rat femoral artery may be mediated by changes in [Ca2+]i through modulation of calcium channel activity.     

Protein kinase C dependent and independent activation of phospholipase A2 under calcium ionophore (A23187) exposure in rabbit pulmonary arterial smooth muscle cells

Exposure of rabbit pulmonary arterial smooth muscle cells to the calcium ionophore A23187, dose-dependently stimulates arachidonic acid (AA) release and phospholipase A2 (PLA2) activity. The protein kinase C (PKC) inhibitor, sphingosine does not prevents AA release and PLA2 activity caused by low doses of A23187. In contrast, sphingosine markedly prevents AA release and PLA2 activity caused by higher doses of A23187. PKC activity profile indicates that treatment of the cells with low doses of A23187 does not cause significant alteration of PKC translocation from cytosol to membrane whereas higher concentrations of the ionophore dose-dependently enhance PKC translocation from cytosol to membrane in the smooth muscle cells.