Regional heterogeneity in the mechanisms of myogenic tone in hamster arterioles

Abstract

Myogenic tone is an important feature of arterioles and resistance arteries, but the mechanisms responsible for this hallmark characteristic remain unclear. We used pharmacological inhibitors to compare the roles played by phospholipase C (PLC; 10 μM U73122), inositol 1,4,5-trisphosphate receptors (IP₃Rs; 100 μM 2-aminoethoxydiphenylborane), protein kinase C (10 μM bisindolylmaleimide I), angiotensin II type 1 receptors (1 μM losartan), Rho kinase (10 nM-30 μM Y27632 or 300 nM H1152), stretch-activated ion channels (10 nM-1 μM Gd³⁺ or 5 μM spider venom toxin GsMTx-4) and L-type voltage-gated Ca²⁺ channels (0.3-100 μM diltiazem) in myogenic tone of cannulated, pressurized (80 cmH₂O), second-order hamster cremaster or cheek pouch arterioles. Effective inhibition of either PLC or IP₃Rs dilated cremaster arterioles, inhibited Ca²⁺ waves, and reduced global Ca²⁺ levels. In contrast, cheek pouch arterioles did not display Ca²⁺ waves and inhibition of PLC or IP₃Rs had no effect on myogenic tone or intracellular Ca²⁺ levels. Inhibition of Rho kinase dilated both cheek pouch and cremaster arterioles with equal efficacy and potency but also reduced intracellular Ca²⁺ signals in both arterioles. Similarly, inhibition of mechanosensitive ion channels with Gd²⁺ or GsMTx-4 produced comparable dilation in both arterioles. Inhibition of L-type Ca²⁺ channels with diltiazem was more effective in dilating cremaster (86 ± 5% dilation, n = 4) than cheek pouch arterioles (54 ± 4% dilation, n = 6, P < 0.05). Thus, there are substantial differences in the mechanisms underlying myogenic tone in hamster cremaster and cheek pouch arterioles. Regional heterogeneity in myogenic mechanisms could provide new targets for drug development to improve regional blood flow in a tissue-specific manner.NEW & NOTEWORTHY Regional heterogeneity in the mechanisms of pressure-induced myogenic tone implies that resistance vessels may be able to alter myogenic signaling pathways to adapt to their environment. A better understanding of the spectrum of myogenic mechanisms could provide new targets to treat diseases that affect resistance artery and arteriolar function.

Keywords: arterioles; hamster; microcirculation; myogenic tone.

Fig. 1.

Phospholipase C (PLC) and inositol 1,4,5-trisphosphate receptors (IP₃Rs) contribute to myogenic tone in cremaster arterioles. Both U73122 and 2-aminoethoxydiphenylborane (2-APB) inhibited myogenic tone in second-order hamster cremaster arterioles. Data are mean diameters ± SE for arterioles before (control), during exposure to U73122 (A; 10 μM, n = 7) or 2-APB (B; 100 μM, n = 5), or during exposure to 0 Ca²⁺ physiological salt solution (PSS) as indicated. *P < 0.05, significantly different from control.

Fig. 2.

PLC and IP₃Rs do not contribute to myogenic tone in cheek pouch arterioles. Neither U73122 nor 2-APB affected myogenic tone in second-order hamster cheek pouch arterioles, but they effectively inhibited phenylephrine-induced constriction. In A and B, data are mean diameters ± SE (n = 6) for arterioles before (control), during exposure to the PLC inhibitor U73122 (A; 10 μM) or 2-APB (B; 100 μM), or during exposure to 0 Ca²⁺ PSS as indicated. C and D: mean diameters ± SE (n = 3) before (PSS) or at the peak of constriction induced by bolus application of 20 nmol phenylephrine as indicated before (control) or during exposure to U73122 (10 μM; C), its nonactive analog U73343 (10 μM; C), or the IP₃R antagonist 2-APB (100 μM; D). *P < 0.05, significantly different from control.

Fig. 3.

Smooth muscle cells in hamster cremaster arterioles display Ca²⁺ waves, whereas cheek pouch arterioles do not. A, inset: image of a tangential, confocal slice through a cremaster arteriole loaded with fluo-4 along with nine regions of interest (ROIs) that were placed at the peak of Ca²⁺ waves. The data plot shows relative fluorescence (F/F_o) for the nine ROIs showing the amplitude and duration of these Ca²⁺ events. Application of SparkAn to these data with a threshold of 15% above baseline revealed 19 Ca²⁺ waves with a mean amplitude of 1.52 ± 0.07 F/F_o (range: 1.2–2.6) during the 16-s recording period shown for this vessel. Similar results were obtained in four additional cremaster arterioles, confirming a previous study (62). B: data similar to those shown in A for a cheek pouch arteriole. In the nine ROIs shown, no events with an amplitude of >15% above baseline were detected during the 16-s recording period shown. Similar results were obtained in five additional cheek pouch arterioles. The scale bars shown in A and B = 20 μm. See text for more details.

Fig. 4.

Lack of effect of U73122 or 2-APB on global Ca²⁺ levels in hamster cheek pouch arterioles. A and B: projected average images of 500 image stacks in the absence (A) and presence (B) of the PLC inhibitor U73122 (10 μM) showing that U73122 does not cause a significant fall in global fluo-4 fluorescence. Scale bars in A and B = 25 μm. C and D: mean fluo-4 intensities ± SE (n = 6) expressed relative to the average fluo-4 intensity in control image stacks before (control) and in the presence of U73122 (10 μM; C) or the IP₃R antagonist 2-APB (100 μM; D). No significant differences were observed (P > 0.05).

Fig. 5.

Lack of effect of the PKC inhibitor bisindolylmaleimide I (BIM I) on the diameter of hamster cremaster arterioles. Data are mean diameters ± SE (n = 5) before (rest) and during exposure to phorbolmysterate acetate (PMA), in the absence (control) and presence of BIM I, or after superfusion with 0 Ca²⁺ PSS as indicated. *P < 0.05, different from control; **P < 0.05, greater than all other groups.

Fig. 6.

Angiotensin II (ANG II) receptors do not contribute to myogenic tone in hamster cremaster arterioles. Effective blockade of ANG II receptors with losartan or α₁-adrenergic receptors with prazosin has no effect on myogenic tone in cremaster arterioles. Data are mean diameters ± SE (n = 5) before (rest) or during exposure to ANG II or phenylephrine (PHE) in the absence (Control) or presence of the ANG II receptor blocker losartan, the α₁-adrenergic receptor blocker prazosin, or exposure to 0 Ca²⁺ PSS as indicated. *P < 0.05, significantly different from rest; **P < 0.05, significantly greater than all other groups.

Fig. 7.

Role of Rho kinase in myogenic tone of cremaster and cheek pouch arterioles. The Rho kinase inhibitor Y27632 causes concentration-dependent dilation of hamster arterioles. Data are mean diameters ± SE (n = 4) in response to cumulative addition of Y27632 to cannulated cremaster or cheek pouch arterioles as indicated. Concentration-dependent dilation was observed in both vessels (P < 0.05). Resting and maximal diameters were 42 ± 2 and 59 ± 1 μm for cremaster arterioles and 40 ± 2 and 58 ± 4 μm for cheek pouch arterioles, respectively. See text for more information.

Fig. 8.

Rho kinase antagonists inhibit myogenic tone and Ca²⁺ signaling in hamster cremaster arterioles. Data are the occurrence of Ca²⁺ waves (Occ; number of cells showing waves/total number of cells studied) ± 95% confidence interval or means ± SE (n = 5) for the amplitude (Amp; F/F_o) or frequency (Freq; Hz) of Ca²⁺ waves and global fluo-4 levels (an index of global Ca²⁺) and arteriolar diameter in the absence (control) or presence of the Rho kinase inhibitors Y27632 (10 μM, n = 5; A) or H-1152 (300 nM, n = 4; B). * P < 0.05, significantly different from control value.

Fig. 9.

Role of mechanosensitive transient receptor potential (TRP) channels in myogenic tone of cremaster and cheek pouch arterioles. Gd³⁺ and the spider venom toxin GsMTx-4 inhibited myogenic tone in hamster arterioles. A: data are mean dilation relative to 0 Ca²⁺ diameter ± SE (n = 4 for cremaster and n = 9 for cheek pouch). The solid curve represents the best fit for a sigmoid (logEC₅₀: −7.26 ± 0.10 and E_max: 0.67 ± 0.04) to both data sets. B: data are mean diameters ± SE (n = 4 for each vessel type) before (rest), in the presence of GsMTx-4, and upon exposure to 0 Ca²⁺ PSS as indicated. *P < 0.05, significantly different from rest; **P < 0.05, significantly greater than all other groups.

Fig. 10.

Diltiazem is more effective in cremaster than cheek pouch arterioles. Data are mean dilation relative to 0 Ca²⁺ PSS diameter ± SE (n = 4 for cremaster and n = 6 for cheek pouch) during cumulative addition of the L-type Ca²⁺ channel blocker diltiazem to the superfusate. Solid curves are the best fit sigmoids. Ditiazem had similar potency (logEC₅₀: −5.99 ± 0.21) in both vessels. However, this Ca²⁺ channel blocker was more effective in cremaster arterioles (E_max: 0.86 ± 0.05) than cheek pouch arterioles (E_max: 0.54 ± 0.04) as indicated. *P < 0.05 for E_max.