Regulation of arterial tone by activation of calcium-dependent potassium channels.

Blood pressure and tissue perfusion are controlled in part by the level of intrinsic (myogenic) vascular tone. However, many of the molecular determinants of this response are unknown. Evidence is now presented that the degree of myogenic tone is regulated in part by the activation of large-conductance calcium-activated potassium channels in arterial smooth muscle. Tetraethylammonium ion (TEA+) and charybdotoxin (CTX), at concentrations that block calcium-activated potassium channels in smooth muscle cells isolated from cerebral arteries, depolarized and constricted pressurized cerebral arteries with myogenic tone. Both TEA+ and CTX had little effect on arteries when intracellular calcium was reduced by lowering intravascular pressure or by blocking calcium channels. Elevation of intravascular pressure through membrane depolarization and an increase in intracellular calcium may activate calcium-activated potassium channels. Thus, these channels may serve as a negative feedback pathway to control the degree of membrane depolarization and vasoconstriction.

Hyperpolarizing vasodilators activate ATP-sensitive K+ channels in arterial smooth muscle.

Vasodilators are used clinically for the treatment of hypertension and heart failure. The effects of some vasodilators seem to be mediated by membrane hyperpolarization. The molecular basis of this hyperpolarization has been investigated by examining the properties of single K+ channels in arterial smooth muscle cells. The presence of adenosine triphosphate (ATP)-sensitive K+ channels in these cells was demonstrated at the single channel level. These channels were opened by the hyperpolarizing vasodilator cromakalim and inhibited by the ATP-sensitive K+ channel blocker glibenclamide. Furthermore, in arterial rings the vasorelaxing actions of the drugs diazoxide, cromakalim, and pinacidil and the hyperpolarizing actions of vasoactive intestinal polypeptide and acetylcholine were blocked by inhibitors of the ATP-sensitive K+ channels, suggesting that all these agents may act through a common pathway in smooth muscle by opening ATP-sensitive K+ channels.

The ATPase activity of subfragment-1 from the hypertrophied heart.

Myosin and subfragment-1 were prepared from rabbit hearts hypertrophied secondary to pulmonary artery constriction. The Ca2+ -stimulated ATPase activity was reduced while the potassium/EDTA-stimulated ATPase activity was unchanged in both the myosin and subfragment 1 (S-1) from hypertrophied hearts. When hypertrophy myosin was mixed with an equal quantity of control myosin, the ATPase activity of the mixed protein fell halfway between control and hypertrophy values. Similar results were obtained with control and hypertrophy S-1. The actin-stimulated ATPase activity of hypertrophy S-1 was slightly depressed but unlike hypertrophy myosin this depression was not significant when compared to normal S-1. This suggests that papain cleavage may have removed part of the conformational difference that exists between control and hypertrophy myosins.

Biochemical and mechanical properties of resistance arteries from normotensive and hypertensive rats.

Microchemical techniques were employed to measure the DNA, contractile proteins, and connective tissue protein composition of 150 micrograms samples of mesenteric and cerebral resistance arteries taken from 25-week-old spontaneously hypertensive (SHR) and control Wistar-Kyoto (WKY) rats. The active and passive mechanical properties of intact resistance arteries also were determined. The DNA content of branches of the posterior cerebral and mesenteric arteries (170 micrometers I.D.) were elevated by nearly 30% in the SHR compared to the WKY. The amounts of actin and myosin when normalized to DNA content were unchanged in SHR mesenteric arteries compared to control, whereas these amounts were decreased by 25% and 49%, respectively, in the SHR cerebral arteries vs control. The functional implications of these contractile protein measurements agreed with determinations of active smooth muscle cell stress-generating capabilities, which were found unchanged in the mesenteric arteries and depressed in the SHR cerebral arteries. Neither the absolute amounts and concentrations (relative to tissue mass) of elastin in mesenteric and cerebral arteries, nor the absolute amounts and concentrations of collagen in the mesenteric artery, were changed in the SHR. However, cerebral artery total collagen was elevated by 31% in the SHR, with no change in collagen concentration between the two strains. Under conditions where the smooth muscle cells were fully relaxed, the internal radii of SHR brain and SHR mesenteric arteries were smaller at all pressures with respect to the WKY. However, only the SHR cerebral arteries were actually less distensible than controls. Thus, it is apparent that hypertension-associated changes in the chemical and mechanical properties of the resistance artery wall vary considerably depending upon which vascular bed is examined. The measurements made in this study suggest that these changes are more pronounced in brain arteries. This finding could be of significance regarding the autoregulatory capability of, and blood pressure distribution within, brain vessels of hypertensive animals.

Endothelium-dependent dilation of feline cerebral arteries: role of membrane potential and cyclic nucleotides.

The objective of this study was to characterize the role of membrane potential and cyclic nucleotides in endothelium-dependent dilation of cerebral arteries. Middle cerebral arteries isolated from cats were depolarized and constricted in response to serotonin or when subjected to transmural pressures greater than 50 mm Hg. Acetylcholine (ACh) and ADP caused vasodilation and a sustained, dose-dependent hyperpolarization of up to 20 mV in this artery. The membrane potential change preceded the vasodilation by approximately 6 s. Hyperpolarizations and dilations to ACh and ADP did not occur in preparations without endothelium. The hyperpolarizations were abolished by ouabain (10(-5) M), which also blocked the dilator response to ACh. However, dilations to ADP were unaffected by ouabain. Methylene blue (5 x 10(-5) M), a guanylate cyclase inhibitor, had no effect on the responses to ACh or ADP in the presence or absence of ouabain. Cyclic guanosine monophosphate (cGMP) levels were not altered in cerebral arteries exposed to ACh or ADP. However, ADP did increase cyclic adenosine monophosphate levels in these blood vessels. We conclude that although membrane hyperpolarizations may be adequate to cause vasodilation, at least one other pathway of endothelium-dependent vasodilation also is present in feline cerebral arteries. Cyclic GMP does not appear to be involved in this alternate pathway of dilation.

Perivascular nerves with immunoreactivity to vasoactive intestinal polypeptide in cephalic arteries of the cat: distribution, possible origins and functional implications.

The distribution of nerves containing vasoactive intestinal polypeptide(VIP)-immunoreactive material was examined in the cephalic arteries and cranial nerves of cats using an indirect immunofluorescence procedure on whole mounts. Perivascular VIP-immunoreactive nerves were widely distributed in arteries and arterioles supplying glands, muscles and mucous membranes of the face. Within the cerebral circulation, perivascular VIP-immunoreactive nerves were most abundant in the circle of Willis and the proximal portions of the major cerebral arteries and their proximal branches supplying the rostral brainstem and ventral areas of the cerebral cortex. Nerves containing VIP-immunoreactive material were absent from distal portions of arteries supplying the posterior brainstem, cerebellum and dorsal cerebral cortex. Cerebral perivascular VIP-immunoreactive nerves had extracerebral origins probably from VIP-immunoreactive perikarya within microganglia in the cavernous plexus and external rete. Extracerebral perivascular VIP-immunoreactive nerves probably arose from VIP-immunoreactive perikarya in microganglia associated with the tympanic plexus, chorda tympani, lingual nerve and Vidian nerve as well as from cells in the otic, sphenopalatine, submandibular and sublingual ganglia. Therefore, it seems likely that each major segment of the cephalic circulation is supplied by local VIP-immunoreactive neurons. If the VIP-immunoreactive nerves cause vasodilation, they are well placed to allow redistribution of arterial blood flow within the head. During heat stress, neurogenic vasodilation of the appropriate beds would permit efficient cooling of cerebral blood, particularly that supplying the rostral brainstem and surrounding areas of the cerebral cortex.

Electrophysiological analysis of neurogenic vasodilatation in the isolated lingual artery of the rabbit.

The nature of neurogenic vasodilatation was investigated in isolated segments of rabbit lingual artery. In separate experiments membrane responses to nerve stimulation were studied by use of microelectrodes. In the presence of guanethidine to block constrictor responses and noradrenaline to induce tone, field stimulation with trains of pulses (8 Hz for 0.5 to 4 s) produced vasodilatation. Atropine (10(-6) M) reduced the relaxations to about 50% of the control values while the induced vasodilatations were potentiated by physostigmine. Tetrodotoxin (TTX, 10(-7) M) blocked all nerve-evoked responses. These data suggest that there is a cholinergic and a non-cholinergic component of the vasodilatation produced by nerve stimulation in the rabbit lingual artery. Single stimuli did not evoke electrophysiological responses. With parameters similar to those used in the mechanical studies, periarterial stimulation in the presence of guanethidine evoked membrane hyperpolarizations which achieved amplitudes of up to 11 mV. The ionophoretic application of acetylcholine (ACh) produced hyperpolarization. The inhibitory junction potentials (i.j.ps) but not the ionophoretic-induced responses were blocked by TTX. The nerve-evoked and the ACh-induced hyperpolarizations were potentiated by physostigmine (5 X 10(-7) M) and totally blocked by atropine (10(-7) M). I.j.ps and hyperpolarization to ionophoresis of ACh were recorded from arteries in which the endothelium had been removed by mechanical rubbing. Mechanical relaxation to field stimulation and ACh was observed in preparations without endothelium. These data suggest that the cholinergic component of the neurogenic vasodilatation in the rabbit lingual artery is accompanied by hyperpolarization. The non-cholinergic component does not appear to possess an electrophysiological correlate. In addition, it seems that the action of nerve-released ACh is mediated by muscarinic receptors which are situated directly on the vascular smooth muscle cells.

Distribution and origins of VIP-immunoreactive nerves in the cephalic circulation of the cat.

VIP-immunoreactive (IR) nerves were visualized in whole mounts and sections of cephalic arteries and cranial nerves of cats with indirect immunofluorescence. Perivascular VIP-IR nerves were very widely distributed in arteries and arterioles supplying glands, muscles and mucous membranes of the face. Within the cerebral circulation, perivascular VIP-IR nerves were most abundant in the Circle of Willis and the proximal portions of the major cerebral arteries and their proximal branches supplying the rostral brain stem and ventral areas of the cerebral cortex. VIP-IR nerves were absent from arterial branches supplying the posterior brain stem, cerebellum and dorsal cerebral cortex. Cerebral perivascular VIP-IR nerves probably arise from VIP-IR perikarya within microganglia found in the cavernous plexus and external rete. Extracerebral perivascular VIP-IR nerves probably arise from VIP-IR perikarya in microganglia associated with the tympanic plexus, chorda tympani, lingual nerve and Vidian nerve as well as from cells in the otic, sphenopalatine, submandibular and sublingual ganglia. It seems likely, therefore, that each major segment of the cephalic circulation is supplied by local VIP-IR neurons.

Neuropeptide Y and vasoactive intestinal polypeptide in cerebral arteries of the rat: relationships between innervation pattern and mechanical response.

Possible relationships between the density of peptide innervation and the contractile response of rat cerebral arteries to exogenously applied neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) were examined. The effects of NPY on membrane potential and reactivity of cerebral arteries to exogenous norepinephrine also were studied. In normally innervated arteries there was no apparent correlation between degree of innervation and response to NPY. Marked, prolonged tachyphylaxis to NPY and VIP was observed following brief exposure to these peptides. Surgical removal of the superior cervical ganglia or the sphenopalatine ganglia greatly reduced and, in some cases, eliminated NPY- or VIP-immunoreactive perivascular nerves from cerebral arteries. However, responses of denervated middle cerebral arteries to exogenous NPY or VIP were not different from responses of innervated arteries. Doses of NPY that induced maximal contraction caused no change in membrane potential of the middle cerebral artery. NPY also did not alter the response of cerebral arteries to exogenous norepinephrine. Finally, electrical stimulation of normal or denervated arteries caused only minor constrictor or dilator responses. These results do not support a substantial role for peptidergic perivascular nerves in regulation of pial arterial contractility in the rat.

Atropine potentiates neurogenic vasodilatation of the feline infraorbital artery: possible mechanisms.

After treatment with guanethidine to inactivate sympathetic nerves, the feline infraorbital artery (IOA) relaxes in response to activation of periarterial nerves in vitro. This response was 60-65% greater in magnitude and 50% longer in duration in the presence of atropine, thus revealing a significant non-adrenergic, non-cholinergic (NANC) dilator response which is potentiated by blockade of muscarinic receptors. Nerve-mediated dilations and the potentiating effect of atropine were endothelial cell-independent. In the presence of atropine the resting membrane potential (-51 +/- 2 mV) of infraorbital vascular smooth muscle cells was not changed by activation of nerves, nor by exogenously applied vasoactive intestinal polypeptide (VIP). Electrical stimulation caused release of VIP from this artery, but atropine did not measurably enhance the degree of release of VIP. Therefore, although presynaptic, muscarinic inhibition of release of a NANC transmitter probably occurs in the IOA, either VIP is not the transmitter involved in this response or the changes in release of VIP are too slight to be detected by the in vitro techniques employed in this study.

Vasoactive intestinal polypeptide and acetylcholine are responsible for neurovasodilation in the head of the cat.

There is reason to believe that the dilator innervation to blood vessels of many of the tissues of the head may be part of a common outflow from the central nervous system. Part of the effect of its activation is due to the release of acetylcholine and evidence is presented that, in addition, vasoactive intestinal polypeptide (VIP) release also plays a role. The non-cholinergic part of the dilator response to nerve activity can in a series of blood vessels be correlated with the level of VIP in their wall and is selectively reduced by VIP antiserum. In addition, VIP has suitable characteristics as a vasodilator. Whereas substance P which is present in the walls of many arteries of the head, does not qualify as a putative dilator transmitter. Despite the fact that the physiological role of dilator innervation to the circulation as a whole is not understood, the powerful consequences of the activation of the system in particular blood vessels suggests that this innervation should be seriously considered when studying the regulation of peripheral resistance.

TRPV channels and vascular function.

Transient receptor potential channels, of the vanilloid subtype (TRPV), act as sensory mediators, being activated by endogenous ligands, heat, mechanical and osmotic stress. Within the vasculature, TRPV channels are expressed in smooth muscle cells, endothelial cells, as well as in peri-vascular nerves. Their varied distribution and polymodal activation properties make them ideally suited to a role in modulating vascular function, perceiving and responding to local environmental changes. In endothelial cells, TRPV1 is activated by endocannabinoids, TRPV3 by dietary agonists and TRPV4 by shear stress, epoxyeicosatrienoic acids (EETs) and downstream of Gq-coupled receptor activation. Upon activation, these channels contribute to vasodilation via nitric oxide, prostacyclin and intermediate/small conductance potassium channel-dependent pathways. In smooth muscle, TRPV4 is activated by endothelial-derived EETs, leading to large conductance potassium channel activation and smooth muscle hyperpolarization. Conversely, smooth muscle TRPV2 channels contribute to global calcium entry and may aid constriction. TRPV1 and TRPV4 are expressed in sensory nerves and can cause vasodilation through calcitonin gene-related peptide and substance P release as well as mediating vascular function via the baroreceptor reflex (TRPV1) or via increasing sympathetic outflow during osmotic stress (TRPV4). Thus, TRPV channels play important roles in the regulation of normal and pathological cellular function in the vasculature.

TRPC3 mediates pyrimidine receptor-induced depolarization of cerebral arteries.

We tested the hypothesis that TRPC3, a member of the canonical transient receptor potential (TRP) family of channels, mediates agonist-induced depolarization of arterial smooth muscle cells (SMCs). In support of this hypothesis, we observed that suppression of arterial SMC TRPC3 expression with antisense oligodeoxynucleotides significantly decreased the depolarization and constriction of intact cerebral arteries in response to UTP. In contrast, depolarization and contraction of SMCs induced by increased intravascular pressure, i.e., myogenic responses, were not altered by TRPC3 suppression. Interestingly, UTP-evoked responses were not affected by suppression of a related TRP channel, TRPC6, which was previously found to be involved in myogenic depolarization and vasoconstriction. In patch-clamp experiments, UTP activated a whole cell current that was greatly reduced or absent in TRPC3 antisense-treated SMCs. These results indicate that TRPC3 mediates UTP-induced depolarization of arterial SMCs and that TRPC3 and TRPC6 may be differentially regulated by receptor activation and mechanical stimulation, respectively.

Membrane hyperpolarization is a mechanism of endothelium-dependent cerebral vasodilation.

Acetylcholine (ACh)-induced hyperpolarization of cerebral arteries requires a functional endothelium. The hyperpolarization is reversed by potassium-channel blockers. The goal of this study was to determine whether the hyperpolarization is causally related to endothelium-dependent dilation of isolated cerebral arteries. ACh hyperpolarized rabbit middle cerebral arteries by up to 19 mV. The hyperpolarizations were sustained and did not occur in arteries without endothelial cells or in the presence of potassium-channel inhibitors (3 x 10(-6) M glibenclamide or 5 x 10(-5) M BaCl2). ACh-induced dilator responses were inhibited but not abolished by glibenclamide or BaCl2. Methylene blue also inhibited the dilator responses, and a combination of glibenclamide or BaCl2 and methylene blue greatly diminished the dilation. Nitric oxide relaxed but did not hyperpolarize the vascular smooth muscle cells, and BaCl2 had no effect on the nitric oxide-induced relaxations. These data indicate that the overall cerebral arterial dilator response to ACh is determined by the combined effects of membrane hyperpolarization, which closes voltage-dependent calcium channels, and the actions of a second endothelial factor, probably endothelium-derived relaxing factor.

Hyperpolarization and relaxation of resistance arteries in response to adenosine diphosphate. Distribution and mechanism of action.

Membrane hyperpolarization appears to be an important mechanism of vasodilation induced by many pharmacological and endogenous vasodilators. The objective of the present study was to determine the mechanisms of vasodilation induced by ADP, and endogenous vasodilator, in various resistance arteries isolated from the rabbit. ADP hyperpolarized (12-15 mV) and relaxed mesenteric and skeletal muscle resistance arteries. The hyperpolarization was abolished by glibenclamide, an inhibitor of ATP-sensitive potassium channels. Glibenclamide inhibited part of the ADP-induced relaxations of these arteries; thus, a portion of the relaxation appears to result directly from the change in membrane potential. Hyperpolarizations and relaxations to low concentrations of ADP (less than 0.3 microM) were abolished by removal of the endothelium, but responses to higher concentrations of ADP were partially independent of the endothelium. ADP did not hyperpolarize but did relax small-diameter middle cerebral arteries, and glibenclamide had no effect on these ADP-induced relaxations. Relaxations of small cerebral arteries to all concentrations of ADP were endothelium dependent. These studies support the hypothesis that activation of ATP-sensitive potassium channels is an important general mechanism of vasodilation, including responses of resistance arteries. However, this generalization may not apply to small pial arteries of the rabbit.

Potassium channels in vascular smooth muscle.

1. Regulation of smooth muscle membrane potential through changes in K+ channel activity and subsequent alterations in the activity of voltage-dependent calcium channels is a major mechanism of vasodilation and vasoconstriction, both in normal and pathophysiological conditions. The contribution of a given K+ channel type to this mechanism of vascular regulation depends on the vascular bed and species examined. 2. Multiple K+ channels are present in most vascular smooth muscle cells and these different K+ channels play unique roles in regulating vascular tone. Voltage-dependent K+ (Kv) channels are activated by depolarization, may contribute to steady state resting membrane potential and are inhibited by certain vasoconstrictors. Calcium-activated K+ (K(Ca)) channels oppose the depolarization associated with intrinsic vascular tone and are activated by some endogenous vasodilators. Small-conductance, apamin-sensitive K(Ca) channels may be activated by endothelium-derived hyperpolarizing factor. ATP-sensitive K+ (K(ATP)) channels are activated by pharmacological and endogenous vasodilators. Inward rectifier K+ (K(ir)) channels are activated by slight changes in extracellular K+ and may contribute to resting membrane potential. 3. Membrane potential and diameter are determined, in part, by the integrated activity of several K+ channels, which are regulated by multiple dilator and constrictor signals in vascular smooth muscle.

Neurogenic muscarinic vasodilation in the cat. An example of endothelial cell-independent cholinergic relaxation.

Nerve-mediated and acetylcholine-induced dilator behavior of feline posterior auricular arteries was studied in vitro. We evaluated the muscarinic nature and endothelial cell-dependence of the vasodilations and attempted to determine if there are inhibitory muscarinic receptors located directly on the smooth muscle cells in this artery. Transmural nerve stimulation of arteries which were pretreated with guanethidine (5 X 10(-6)M) and constricted with prostaglandin F2 alpha (3 X 10(-6)M) caused a frequency-dependent, tetrodotoxin-sensitive relaxation of up to 50% of induced tone. Atropine (10(-7)M) blocked more than 95% of this response at all frequencies. Removal of the endothelium by rubbing the intimal surface did not affect the magnitude of the response, but prolonged it slightly. Neurogenic relaxations in rubbed preparations were atropine-sensitive, although less so than control at higher stimulation frequencies. Relaxation of this artery to the calcium ionophore A23187 was completely endothelial cell-dependent. However, exogenous acetylcholine caused dose-dependent relaxations both in control and rubbed preparations. We conclude that the posterior auricular artery is an example of a blood vessel which has muscarinic receptors located directly on its smooth muscle cells which, when activated by acetylcholine released from perivascular nerves, mediate a smooth muscle cell relaxation. This finding contrasts with models of the vascular smooth muscle cell which indicates an excitatory role for muscarinic receptors.

Regulation of arterial tone by calcium-dependent K+ channels and ATP-sensitive K+ channels.

Resistance arteries depolarize and constrict to elevations in intravascular pressure. However, many of the molecular aspects of this phenomenon are not known. We present evidence that large conductance calcium-dependent potassium (KCa) channels, which are activated by intracellular calcium and membrane depolarization, play a fundamental role in regulating the degree of intravascular pressure-induced, myogenic tone. We found that blockers of KCa channels, charybdotoxin (CTX, < 100 nM) and TEA+ (< 0.5 mM), further depolarized pressurized arteries by as much as 12 mV and decreased diameter by up to 40%. CTX blocked KCa channels in outside-out patches from arterial smooth muscles with half-block constant of 10 nM and external TEA+ caused a flickery block, with a half-block constant of 200 microM. We propose that KCa channels serve as a negative feedback pathway to limit the degree of membrane depolarization and hence vasoconstriction to pressure. In contrast, CTX and TEA+ (< 1 mM) were without effect on membrane hyperpolarization and dilation to a wide variety of synthetic (cromakalim, pinacidil, diazoxide, minoxidil sulfate) and endogenous agents [calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide, an endothelial-derived hyperpolarizing factor]. Glibenclamide and low concentrations of external barium that inhibit ATP-sensitive potassium (KATP) channels, however, blocked the hyperpolarizations and dilations to these substances. We have identified KATP channels as well as high-affinity glibenclamide binding sites in arterial smooth muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that vasoactive intestinal polypeptide (VIP) mediates neurogenic vasodilation of feline cerebral arteries.

In this study the magnitude of non-sympathetic, non-cholinergic neurogenic vasodilation of feline cerebral arteries in vitro was correlated with the extent of innervation by VIP-immunoreactive nerves. Well-innervated arteries underwent nerve-mediated relaxation whereas those that are not supplied with VIP-containing axons did not relax to transmural nerve stimulation. The relaxation of cerebral arteries that are well endowed with VIP-immunoreactive nerves was selectively and reversibly inhibited by VIP-specific antiserum. Substance P-specific antiserum did not affect the dilator responses. We conclude that VIP is a functional neurodilator transmitter in the cerebral circulation.