Conducted Vasomotor Responses Research Articles

Reduced tubuloglomerular feedback activity and absence of its synchronization in a connexin40 knockout rat.

Introduction: Tubuloglomerular feedback (TGF) is the negative feedback component of renal blood flow (RBF) autoregulation. Neighbouring nephrons often exhibit spontaneous TGF oscillation and synchronization mediated by endothelial communication, largely via connexin40 (Cx40). Methods: We had a knockout (KO) rat made that lacks Cx40. One base pair was altered to create a stop codon in exon 1 of Gja5, the gene that encodes Cx40 (the strain is WKY-Gja55em1Mcwi). Blood pressure (BP)-RBF transfer functions probed RBF dynamics and laser speckle imaging interrogated the dynamics of multiple efferent arterioles that reach the surface (star vessels). Results: The distribution of wild type (WT), heterozygote, and KO pups at weaning approximated the Mendelian ratio of 1:2:1; growth did not differ among the three strains. The KO rats were hypertensive. BP-RBF transfer functions showed low gain of the myogenic mechanism and a smaller TGF resonance peak in KO than in WT rats. Laser speckle imaging showed that myogenic mechanism had higher frequency in KO than in WT rats, but similar maximum spectral power. In contrast, the TGF frequency was similar while peak power of its oscillation was much smaller in KO than in WT rats. In WT rats, plots of instantaneous TGF phase revealed BP-independent TGF synchronization among star vessels. The synchronization could be both prolonged and widespread. In KO rats TGF synchronization was not seen, although BP transients could elicit short-lived TGF entrainment. Discussion: Despite the reduced TGF spectral power in KO rats, there was sufficient TGF gain to induce oscillations and therefore enough gain to be effective locally. We conclude that failure to synchronize is dependent, at least in part, on impaired conducted vasomotor responses.

Conceptualizing conduction as a pliant electrical response: impact of gap junctions and ion channels.

Vasomotor responses conduct among resistance arteries to coordinate blood flow delivery pursuant to energetic demand. Conduction is set by the electrical and mechanical properties of vascular cells, the former tied to how gap junctions and ion channels distribute and dissipate charge, respectively. These membrane proteins are subject to modulation; thus, conduction could be viewed as "pliant" to the current regulatory state. This study used in silico approaches to conceptualize electrical pliancy and to illustrate how gap junctional and ion channel properties distinctly impact conduction along a single skeletal muscle artery or a branching cerebrovascular network. Initial simulations revealed how vascular cells encoded with electrotonic properties best reproduced spreading behavior; the endothelium's importance as a charge source and a longitudinal conduit was readily observed. Alterations in gap junctional conductance produced unique electrical fingerprints: 1) decreased endothelial coupling impaired longitudinal but enhanced radial spread, and 2) reduced myoendothelial coupling limited radial but enhanced longitudinal spread. Subsequent simulations illustrated how tuning ion channel activity, e.g., inward rectifying- and voltage-gated K+ channels, modified charge dissipation, resting membrane potential, and the spread of the electrical phenomenon. Restricting ion channel tuning to a network subregion then revealed how electrical spread could be locally shaped in accordance with the aggregate changes in membrane resistance. In summary, our analysis frames and reimagines electrical conduction as a pliable process, with subtle regulatory changes to membrane proteins shaping network spread and tissue perfusion.NEW & NOTEWORTHY Conducted vasomotor responses depend on initiation and spread of electrical phenomena along arterial walls and their translation into contractile responses. Using computational approaches, we show how subtle but widespread regulation of gap junctions and ion channels can modulate the range and amplitude of electrical spread. Ion channels are regulated by endocrine and mechanical signals and may differ regionally in networks. Subregional electrical changes are not spatially confined but may affect electrical conduction in neighboring regions.

Origins of variation in conducted vasomotor responses.

Regulation of blood flow in the microcirculation depends on synchronized vasomotor responses. The vascular conducted response is a synchronous dilatation or constriction, elicited by a local electrical event that spreads along the vessel wall. Despite the underlying electrical nature, however, the efficacy of conducted responses varies significantly between different initiating stimuli within the same vascular bed as well as between different vascular beds following the same stimulus. The differences have stimulated proposals of different mechanisms to account for the experimentally observed variation. Using a computational approach that allows for introduction of structural and electrophysiological heterogeneity, we systematically tested variations in both arteriolar electrophysiology and modes of stimuli. Within the same vessel, our simulations show that conduction efficacy is influenced by the type of cell being stimulated and, in case of depolarization, by the stimulation strength. Particularly, simultaneous stimulation of both endothelial and vascular smooth muscle cells augments conduction. Between vessels, the specific electrophysiology determines membrane resistance and conduction efficiency-notably depolarization or radial currents reduce electrical spread. Random cell-cell variation, ubiquitous in biological systems, only cause small or no reduction in conduction efficiency. Collectively, our simulations can explain why CVRs from hyperpolarizing stimuli tend to conduct longer than CVRs from depolarizing stimuli and why agonists like acetylcholine induce CVRs that tend to conduct longer than electrical injections. The findings demonstrate that although substantial heterogeneity is observed in conducted responses, it can be largely ascribed to the origin of electrical stimulus combined with the specific electrophysiological properties of the arteriole. We conclude by outlining a set of "principles of electrical conduction" in the microcirculation.

Tuning electrical conduction along endothelial cell tubes via Ca2+‐activated K+ channels

Electrical conduction along coupled cells reflects low axial resistance (ra) of gap junction channels (GJCs) and high plasma membrane resistance (rm) to current flow. Manipulating GJCs alters conducted vasomotor responses but little is known of how conduction may be governed by membrane ion channel activation. We hypothesized that Ca2+‐activated K+ channels (SKCa/IKCa) can tune rm to regulate electrical conduction along endothelial cell (EC) tubes (width, 60 μm; length, ≥ 2 mm) isolated from feed arteries of C57BL/6 mouse skeletal muscle (n = 6–10 males). Using dual simultaneous intracellular microelectrodes, membrane potential (Vm) was uniform along EC tubes (−25 ± 2 mV) and propidium iodide dye injected into one EC spread throughout neighbouring ECs. Conduction amplitude (CA = ΔVm at Site 2/nA current injected at Site 1) decreased with electrode separation distance (CA = 11, 8, 6, 4, 3 ± 1 mV/nA at 50, 500, 1000, 1500, 2000 μm). The SKCa/IKCa activator NS309 (1μM) reduced length constant [λ = (rm/ra)½] from 1360 ± 80 to 840 ± 60 μm (P<0.05). With electrode separation = 500 μm, NS309 from 0.01 to 10 μM increased Vm from −28 ± 1 to −81 ± 1 mV and abolished CA while dye transfer was maintained; blocking SKCa/IKCa (300 nM apamin + 100 nM charybdotoxin) increased CA by ~30% (P<0.05). We conclude that the effectiveness of electrical conduction can be tuned through SKCa/IKCa activation. (NIH R37HL041026, R01HL086483, F32HL110701)

BKCa and KV channels limit conducted vasomotor responses in rat mesenteric terminal arterioles

Intracellular Ca(2+) signals underlying conducted vasoconstriction to local application of a brief depolarizing KCl stimulus was investigated in rat mesenteric terminal arterioles (<40μm). Using a computer model of an arteriole segment comprised of coupled endothelial cells (EC) and vascular smooth muscle cells (VSMC) simulations of both membrane potential and intracellular [Ca(2+)] were performed. The "characteristic" length constant, λ, was approximated using a modified cable equation in both experiments and simulations. We hypothesized that K(+) conductance in the arteriolar wall limit the electrotonic spread of a local depolarization along arterioles by current dissipation across the VSMC plasma membrane. Thus, we anticipated an increased λ by inhibition of voltage-activated K(+) channels. Application of the BK(Ca) channel blocker iberiotoxin (100nM) onto mesenteric arterioles in vitro and inhibition of BK(Ca) channel current in silico increased λ by 34% and 32%, respectively. Similarly, inhibition of K(V) channels in vitro (4-aminopyridine, 1mM) or in silico increased λ by 41% and 21%, respectively. Immunofluorescence microscopy demonstrated expression of BK(Ca), Kv1.5, Kv2.1, but not Kv1.2, in VSMCs of rat mesenteric terminal arterioles. Our results demonstrate that inhibition of voltage-activated K(+) channels enhance vascular-conducted responses to local depolarization in terminal arterioles by increasing the membrane resistance of VSMCs. These data contribute to our understanding of how differential expression patterns of voltage-activated K(+) channels may influence conducted vasoconstriction in small arteriolar networks. This finding is potentially relevant to understanding the compromised microcirculatory blood flow in systemic vascular diseases such as diabetes mellitus and hypertension.

Conducted vasodilatation in human coronary arterioles

Conducted vasomotor responses in the coronary microcirculation match blood flow to metabolic requirements. Here we established whether conducted vasodilatation occurs in human coronary arterioles. Coronary arterioles were dissected from atrial appendage of patients (n=15). In isolated, pressurized arterioles (~ 100 μm) conducted dilatation was initiated by locally administering bradykinin (BK, 100 μM) ejected from a micropipette, positioned in close proximity to the vessel wall. Diameter changes were measured at local and upstream sites (500 and 1,000 μm from the stimulus) with videomicroscopy. Local administration of BK elicited vasodilatation (90±2%), which remained substantial at the upstream sites (500 μm, 53±7%; 1,000 μm, 46±9%). Administration of the gap junction uncoupler carbenoxolone (10 μM) did not affect local responses, but diminished conducted dilatation. The NO synthase inhibitor, L‐NAME had no affect on local responses (82±5%), but significantly reduced dilatation at upstream sites (500 μm, 17±3%; 1,000 μm, 24±6%). Both local and upstream dilatations to BK were diminished by Ca‐activated K‐channel blockers, TRAM34 and apamin and also with 30 mM K+. Thus, BK‐induced dilatation is propagated along the isolated human coronary arteriole. The conducted response is mediated by gap junctional hyperpolarisation spread, which is facilitated by NO. (NIH HL104126, BHF RE/08/004)

Connexin abundance in resistance vessels from the renal microcirculation in normo- and hypertensive rats

The expression of connexins in renal arterioles is believed to have a profound impact on conducted responses, regulation of arteriolar tonus and renal blood flow. We have previously shown that in renal preglomerular arterioles, conducted vasomotor responses are 40% greater in spontaneously hypertensive rats (SHR) than in normotensive Sprague–Dawley (SD) rats. Because conducted vasomotor responses depend on the cell–cell communication mediated through gap junctions, we hypothesized that the increased magnitude of conducted vasomotor response in SHR is associated with an increased amount of connexins in renal arterioles. To test this hypothesis, the amount of connexin 37 (Cx37), Cx40 and Cx43 was assessed in renal arterioles from normo- and hypertensive rats using quantitative immunofluorescence laser confocal miscroscopy. To account for differences in genetic background, we included both normotensive Wistar–Kyoto (WKY) and SD rats in the study. In all three strains of rats, and for all three isoforms, the expression of connexins was predominantly confined to the endothelial cells. We found a significantly increased abundance (240 ± 17.6%, p<0.05) of Cx37 in arterioles from WKY compared with SD and SHR. This high abundance of Cx37 was not related to blood pressure because normotensive SD demonstrated a level of Cx37 similar to that of SHR. Additionally, we found no evidence for an increased abundance of Cx40 and Cx43 in renal arterioles of SHR when compared with normotensive counterparts.

Oxygen sensing and conducted vasomotor responses in mouse cremaster arterioles in situ

This study examines mechanisms by which changes in tissue oxygen tension elicit vasomotor responses and whether localized changes in oxygen tension initiates conducted vasomotor responses in mouse cremaster arterioles. Intravital microscopy was used to visualize the mouse cremaster microcirculation. The cremaster was superfused with Krebs' solution with different oxygen tensions, and a gas exchange chamber was used to induce localized changes in oxygen tension. In arterioles where red blood cells were removed by buffer perfusion, arterioles responded with same magnitudes of vasodilatation (DeltaD = 16.0 +/- 4.9 microm) when changing from high (PO(2) = 242.5 +/- 13.3 mm Hg) to low (PO(2) = 22.5 +/- 4.8 mm Hg) oxygen tension as seen in the intact cremaster circulation (DeltaD = 18.7 +/- 1.0 microm). Blockade of NO synthases by L: -NAME and adenosine receptors by DPCPX had no effects on vasomotor responses to low or high oxygen. Induction of localized low (PO(2) = 23.3 +/- 5.7 mmHg) or high (PO(2) = 300.0 +/- 25.7 mm Hg) oxygen tension caused vasodilatation or -constriction locally and at a site 1,000 microm upstream (distantly). Glibenclamide blocker of ATP-sensitive K(+) channels inhibited vasodilatation and -constriction to low (PO(2) = 16.0 +/- 6.4 mm Hg) and high (PO(2) = 337.4 +/- 12.8 mm Hg) oxygen tension. 1) ATP-sensitive K(+) channels seem to mediate, at least in part, vasodilatation and vasoconstriction to low and high oxygen tension; 2) Red blood cells are not necessary for inducing vasodilatation and vasoconstriction to low or high oxygen tension; 3) localized changes in the oxygen tension cause vasomotor responses, which are conducted upstream along arterioles in mouse cremaster microcirculation.

Connexins and gap junctions in the EDHF phenomenon and conducted vasomotor responses

It is becoming increasingly evident that electrical signaling via gap junctions plays a central role in the physiological control of vascular tone via two related mechanisms (1) the endothelium-derived hyperpolarizing factor (EDHF) phenomenon, in which radial transmission of hyperpolarization from the endothelium to subjacent smooth muscle promotes relaxation, and (2) responses that propagate longitudinally, in which electrical signaling within the intimal and medial layers of the arteriolar wall orchestrates mechanical behavior over biologically large distances. In the EDHF phenomenon, the transmitted endothelial hyperpolarization is initiated by the activation of Ca(2+)-activated K(+) channels channels by InsP(3)-induced Ca(2+) release from the endoplasmic reticulum and/or store-operated Ca(2+) entry triggered by the depletion of such stores. Pharmacological inhibitors of direct cell-cell coupling may thus attenuate EDHF-type smooth muscle hyperpolarizations and relaxations, confirming the participation of electrotonic signaling via myoendothelial and homocellular smooth muscle gap junctions. In contrast to isolated vessels, surprisingly little experimental evidence argues in favor of myoendothelial coupling acting as the EDHF mechanism in arterioles in vivo. However, it now seems established that the endothelium plays the leading role in the spatial propagation of arteriolar responses and that these involve poorly understood regenerative mechanisms. The present review will focus on the complex interactions between the diverse cellular signaling mechanisms that contribute to these phenomena.

Connexin abundance in resistance vessels from the renal microcirculation in normo‐ and hypertensive rats

The expression of connexins in renal arterioles is believed to have a profound impact on conducted responses, regulation of arteriolar tonus and renal blood flow. We have previously shown that in renal preglomerular arterioles, conducted vasomotor responses are 40% greater in spontaneously hypertensive rats (SHR) than in normotensive Sprague-Dawley (SD) rats. Because conducted vasomotor responses depend on the cell-cell communication mediated through gap junctions, we hypothesized that the increased magnitude of conducted vasomotor response in SHR is associated with an increased amount of connexins in renal arterioles. To test this hypothesis, the amount of connexin 37 (Cx37), Cx40 and Cx43 was assessed in renal arterioles from normo- and hypertensive rats using quantitative immunofluorescence laser confocal microscopy. To account for differences in genetic background, we included both normotensive Wistar-Kyoto (WKY) and SD rats in the study. In all three strains of rats, and for all three isoforms, the expression of connexins was predominantly confined to the endothelial cells. We found a significantly increased abundance (240 +/- 17.6%, p<0.05) of Cx37 in arterioles from WKY compared with SD and SHR. This high abundance of Cx37 was not related to blood pressure because normotensive SD demonstrated a level of Cx37 similar to that of SHR. Additionally, we found no evidence for an increased abundance of Cx40 and Cx43 in renal arterioles of SHR when compared with normotensive counterparts.

Mechanism of ATP-Induced Local and Conducted Vasomotor Responses in Isolated Rat Cerebral Penetrating Arterioles

Background: Adenosine triphosphate (ATP), a potent vascular regulator in the cerebral circulation, initiates conducted vasomotor responses which may be impaired after pathological insults. We analyzed the mechanism of ATP-induced local vasomotor responses and their effect on conducted vasomotor responses in rat cerebral penetrating arterioles. Methods: Arterioles were cannulated and their internal diameter monitored. Vasomotor responses to ATP were observed in the presence or absence of inhibitors, or after endothelial impairment. Smooth muscle membrane potentials were measured in some vessels. Results: Microapplication of ATP produced a biphasic response (constriction followed by dilation), which resulted in conducted dilation preceded by a membrane hyperpolarization. α,β-methylene-ATP or pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) blunted the ATP-mediated constriction and enhanced local and conducted dilation. N^ω-monomethyl-L-arginine, endothelial impairment and N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH) reduced the local dilation caused by ATP. The conducted dilation was attenuated by MS-PPOH and endothelial impairment, but not N^ω-monomethyl-L-arginine or indomethacin. Conclusion: ATP-induced conducted dilation is preceded by membrane hyperpolarization. Local ATP induces initial local constriction via smooth-muscle P_2X1 and subsequent dilation via endothelial P_2Y receptors. Nitric oxide, cytochrome P450 metabolites, and intermediate and large conductance K_Ca channels mediate dilation caused by ATP. ATP-induced conducted dilation is dependent upon both the endothelium and cytochrome P450 metabolites.

Postischemic Augmentation of Conducted Dilation in Cerebral Arterioles

Conducted vasomotor responses likely play an important role in cerebrovascular regulation, but it is unclear how these responses may be affected by ischemia. The purpose of this study was to evaluate the hypothesis that cerebral ischemia and reperfusion (I/R) alters vascular conduction in cerebral arterioles. Middle cerebral artery occlusion (MCAO) was induced by an intraluminal filament technique in 4 groups of rats: (A) 2-hour MCAO/24-hour reperfusion (n=14); (B) 2-hour MCAO/1-hour reperfusion (n=7); (C) 1-hour MCAO/24-hour reperfusion (n=6); and (D) 1-hour MCAO/1-hour reperfusion (n=5). Neurological status and infarction (2,3,5-triphenyltetrazolium chloride staining) were evaluated after I/R. Conducted vasomotor responses were assessed in intracerebral branches of the MCA, by following the longitudinal spread of vasodilation or vasoconstriction to localized microapplication of ATP or adenosine. Local microapplication of ATP evoked a biphasic constriction (17+/-3%) and dilation (7+/-2%) response, whereas adenosine elicited only dilation (11+/-2%). These local responses spread longitudinally along sham-control arterioles (1 mm conduction distance) with rapid spatial decay. Ischemia followed by 24-hour reperfusion (groups A and C) led to a marked potentiation of conducted dilation responses: dilation to ATP conducted with virtually no decay in I/R arterioles. Augmentation of conductivity was not observed in the 1-hour reperfusion groups (B and D). Moreover, I/R did not alter conducted constriction. Ischemia-reperfusion led to a specific augmentation of conducted vasodilation in cerebral arterioles. Presumably, enhanced conductivity may improve cerebral perfusion after ischemia.

ATP‐induced local and conducted vasomotor mechanisms in isolated rat cerebral penetrating arterioles

Background ATP is a regulator in the cerebral circulation and initiates conducted vasomotor responses. We analyzed the mechanism of ATP-induced local and conducted vasomotor responses in rat cerebral penetrating arterioles. Methods Arterioles were cannulated and their internal diameter monitored with a video microscope. Vasomotor responses to micro-application of ATP were obtained in the presence or absence of inhibitors. Intracellular membrane potentials of smooth muscle cells were measured in some vessels. Results Topical micro-application of ATP produced a biphasic response (constriction followed by dilation), resulting in conducted vasomotor responses. Conducted dilation was preceded by membrane hyperpolarization. a.b-methylene-ATP or pyridoxal phosphate-6-azophenyl-2′,4′-disulphonic acid blunted the ATP-mediated constriction and enhanced the dilation. No-monomethyl-L-arginine, clotrimazole, and endothelial impairment inhibited the local dilation of ATP. In contrast, the conducted dilation was attenuated by clotrimazole, MS-PPOH and endothelial impairment, but not No-monomethyl-L-arginine. Conclusions Our results indicate that 1) ATP-induced conducted dilation is preceded by hyperpolarization; 2) ATP induces initial local constriction via smooth muscle P2X1 and secondary dilation via endothelial P2Y2 receptors in the cerebral arterioles; 3) Nitric oxide and cytochrome P-450 metabolites contribute to local dilation of ATP; 4) ATP-induced conducted dilation is regulated by the endothelium and cytochrome P-450 metabolites. NIH HL57540 (HHD) and NS30555 (RGD and HHD).

Effects of middle cerebral artery occlusion on the conduction of vasomotor responses along rat cerebral arterioles

Vascular communication mechanisms, such as conducted vasomotor responses, likely play an important role in cerebrovascular regulation, but it is unclear how these mechanisms may be affected by ischemia. In this study, we determined the effect of focal cerebral ischemia on the conducted vasomotor responses of intracerebral arterioles to ATP and to adenosine (Ado). After 2- hours of middle cerebral artery occlusion (MCAO) and a 24-hour reperfusion period in Sprague Dawley rats, intracerebral branches of the MCA were isolated and cannulated with a concentric micropipette system. Both ischemic (n=13) and sham-control (n=8) arterioles developed similar myogenic tone (~70% of passive diameter, 60 mmHg). Pressure-pulse application of Ado (10 mM) and ATP (5 mM) induced both a direct local response, and a secondary conducted response that spread longitudinally along the vessel. ATP caused a local biphasic response of constriction (17±2%) followed by dilation (7±3%), whereas Ado elicited only dilation (13±2%). In sham-control vessels, the constriction/dilation responses to ATP markedly decreased (71%/57%) over a 1 mm distance. Ado-induced dilation decayed even more rapidly and was undetectable at 1 mm. In ischemic vessels, the decay of ATP-induced constriction (70%) was similar to that in sham-controls. On the other hand, dilation responses to either ATP or Ado conducted with virtually no decay in ischemic arterioles over the same distance. We conclude that ischemia-reperfusion led to a specific upregulation of conducted vasodilaton in rat cerebral arterioles. Funded by AHA Grant 0255703N.

Connexin45 (Cx45) cannot replace the function of Cx40 in arterioles

Intercellular communication via gap junction channels (GJC) contributes to the coordination of arteriolar tone which is reflected by conducted vasomotor responses. GJC in arterioles are composed of Cx40, 37, 45 and 43. It is unknown if Cx are interchangeable or exhibit specific properties to support cellular function. We investigated if Cx45, which exhibits a smaller conductance than Cx40, can functionally replace Cx40 using mice in which Cx45 is expressed in place of Cx40 (Cx40KI45). Propagation of dilations and constrictions initiated by acetylcholine (ACh, 1mM) or KCl (3M) were studied in arterioles (≈33μm) of the cremaster muscle by intravital microscopy in Cx40-deficient (Cx40KO), Cx40KI45, and wildtype (wt) mice. ACh and KCl were applied with micropipettes in a confined manner. ACh induced a similar dilation in all genotypes at the site of stimulation (58-69%). This dilation was conducted to distant sites and the amplitude was 54±8% at 1200μm upstream in wt. However, in Cx40KO this remote dilation was significantly reduced to 29±4%. In Cx40KI45 remote dilations were impaired to a similar degree (21±4%). In contrast, the propagation of KCl-induced constrictions was comparable in all genotypes (local: −52 to −65%, 600μm:−21 to −25%). In summary, Cx40 function is critical for the conduction of dilations and cannot be compensated for by Cx45 substitution. Therefore, it appears that specific Cx-properties are necessary to support conduction along the endothelium.

Double knockout of connexins discloses myoendothelial cell communication in mouse cremasteric arterioles.

We utilized homologous recombination to produce conditional endothelial cell-specific knockout of connexin 43 (EC KO), global knockout of connexin 40 (40 KO) and a combination of the two deletions (double KO) in mice. Reactivity to phenylephrine (PE), KCL, and acetylcholine (ACh) were assessed using micropipette application of the agents. Responses were compared at the site of application (local) and at 500 and 1000 mm distant. The response to PE in WT, Cx40 KO and EC KO animals consisted of a rapid arteriolar contraction that peaked within 4 sec and then returned to baseline over approximately 40 sec. In response to PE, arterioles in the double KO animal manifested an extended (recovery > 1 min) and larger local response, and a proportional increase in the magnitudes of the conducted vasomotor responses. Conducted responses at 500 and 1000 μm were similar, suggesting that the longitudinal communication pathway was relatively intact in spite of the manipulation of connexins 40 and 43. The arteriolar responses to PE in the double knockout animals are similar to responses previously reported when endothelial cell Ca++changes in response to PE were buffered using BAPTA (Yashiro and Duling, Circ. Res. 87:1048, 2000), and we therefore hypothesize that deletion of the two connexins eliminates some aspect of cell-cell communication. Supported by USPHS Grant # HL53318

The Conduction of Dilation along an Arteriole Is Diminished in the Cremaster Muscle of Hypertensive Hamsters

Arteriolar vasomotor responses can include a component that conducts along the vessel through gap junction channels. This study examined conducted vasomotor responses in arterioles of the hypertensive hamster. The cremaster muscle of normotensive (CHF-148) and spontaneously hypertensive (CHF-H4) hamsters was exteriorized. Micropipettes containing phenylephrine (0.1 M) or acetylcholine (ACh; 1.0 M) were positioned along second-order arterioles and diameter responses were recorded locally for every 0.4 mm upstream to 1.6 mm. Substantative local constrictions to phenylephrine(PE) were poorly conducted to the 0.4-mm site in normotensive and hypertensive hamsters. Local dilation to ACh decayed by 3 ± 1 µm/mm as it conducted along arterioles of the normotensive hamster. In contrast, conducted dilation decayed by 7 ± 1 µm/mm (p < 0.05) in the hypertensive hamster. This hypertension-induced increase in decay was reversed by α-adrenergic receptor blockade (phentolamine: 1 µM). However, arteriolar constriction to global α₁- (PE) and α₂- (clonidine) adrenergic agonists was unaffected by hypertension. Rather, sympathetic nervous activity was elevated in the hypertensive hamster as indicated by a greater reduction in arterial pressure upon sympathetic ablation (hexamethonium infusion: 30 mg/kg). This study provides the first evidence that vascular cell-cell communication is altered by the elevated sympathetic nervous activity observed in the hypertensive hamster.

Endothelial cell signaling during conducted vasomotor responses.

ACh and KCl stimulate vasomotor responses that spread rapidly and bidirectionally along arteriole walls, most likely via spread of electric current or Ca2+ through gap junctions. We examined these possibilities with isolated, cannulated, and perfused hamster cheek pouch arterioles (50- to 80-microm resting diameter). After intraluminal loading of 2 microM fluo 3 to measure Ca2+ or 1 microM di-8-ANEPPS to measure membrane potential, photometric techniques were used to selectively measure changes in intracellular Ca2+ concentration ([Ca2+]i) or membrane potential in endothelial cells. Activation of the endothelium by micropipette application of ACh (10-4 M, 1.0-s pulse) to a short segment of arteriole (100-200 microm) increased endothelial cell [Ca2+]i and caused hyperpolarization at the site of stimulation. This response was followed rapidly by vasodilation of the entire arteriole ( approximately 2-mm length). Change in membrane potential always preceded dilation, both at the site of stimulation and at distant sites along the arteriole. In contrast, an increase in endothelial cell [Ca2+]i was observed only at the application site. Micropipette application of KCl, which can depolarize both smooth muscle and endothelial cells (250 mM, 2.5-s pulse), also caused a rapid, spreading response consisting of depolarization followed by vasoconstriction. With KCl stimulation, in addition to changes in membrane potential, increases in endothelial cell [Ca2+]i were observed at distant sites not directly exposed to KCl. The rapid longitudinal spread of both hyperpolarizing and depolarizing responses support electrical coupling as the mode of signal transmission along the arteriolar length. In addition, the relatively short distance between heterologous cell types enables the superimposed radial Ca2+ signaling between smooth muscle and endothelial cells to modulate vasomotor responses.

Participation of intracellular Ca2+ stores in arteriolar conducted responses.

We examined the role played by intracellular Ca2+ stores in conducted vasomotor responses induced by phenylephrine (PE) in isolated hamster cremasteric arterioles. When applied briefly ( approximately 1 s) to isolated, cannulated arterioles by using pressure-pulse ejection from a micropipette, PE produced a strong local vasoconstriction and a very small biphasic conducted response (a small constriction followed by a dilation) that propagated several hundred micrometers along the vessel length. The conducted vasomotion was associated with a monophasic elevation of the endothelial cell intracellular Ca2+ concentration ([Ca2+]i) at the site of stimulation, as measured with the Ca2+ indicator fura 2. The Ca2+ pump inhibitor thapsigargin was used to limit filling of Ca2+ stores in smooth muscle and endothelial cells. Thapsigargin reduced baseline diameter and elicited a strong dilator component at the local site while enhancing both the constrictor and dilator components of the PE-induced conducted response. The enhanced conducted constrictor component induced by thapsigargin was mimicked by extraluminal application of tetraethylammonium or charybdotoxin but not by iberiotoxin, apamin, glibenclamide, barium, or 4-aminopirydine. Thapsigargin increased the estimated basal endothelial cell [Ca2+]i by approximately 60 nM and converted the PE-induced change in [Ca2+]i from monotonic to biphasic with a late elevation of [Ca2+]i above baseline that coincided with the increased dilatory component of the conducted response. Luminal application of charybdotoxin plus apamin significantly reduced the dilatory component of the conducted response. These results indicate that intracellular Ca2+ stores play a dynamic role in regulating conducted vasomotor responses apparently through modulation of KCa channels in both cell types.

Expression of connexin 37, 40 and 43 in rat mesenteric arterioles and resistance arteries.

Connexins are the protein constituents of gap junctions which mediate intercellular communication in most tissues. In arterioles gap junctions appear to be important for conduction of vasomotor responses along the vessel. Studies of the expression pattern of connexin isoforms in the microcirculation are sparse. We investigated the expression of the three major vascular connexins in mesenteric arterioles (diameter <50 micro m) from male Sprague-Dawley rats, since conducted vasomotor responses have been described in these vessels. The findings were compared with those obtained from upstream small resistance arteries. Indirect immunofluorescence techniques were used on whole mounts of mesenteric arterioles and on frozen sections of resistance arteries (diameter approximately 300 micro m). Mesenteric arterioles expressed Cx40 and Cx43 in the endothelial layer, and Cx37 was found in most but not all vessels. Connexins were not demonstrated in the media. In resistance arteries endothelial cells expressed Cx37, Cx40 and Cx43. Ultrastructural studies of mesenteric arterioles confirmed that gap junction plaques between endothelial cells are present, whereas myoendothelial, or smooth muscle cell gap junctions could not be demonstrated. The findings suggest that smooth muscle cells in mesenteric arterioles may not be well coupled and favour that conducted vasomotor responses in these vessels are propagated through the endothelial cell layer.