Dissection of two Cx37-independent conducted vasodilator mechanisms by deletion of Cx40: electrotonic versus regenerative conduction

Abstract

Conduction of changes in diameter plays an important role in the coordination of peripheral vascular resistance and, thereby, in the control of arterial blood pressure. It is thought that conduction of vasomotor signals relies on the electrotonic spread of changes in membrane potential from a site of stimulation through gap junctions connecting the cells of the vessel wall. To explore this idea, we stimulated a short segment of mouse cremasteric arterioles with an application, via micropipette, of ACh, an endothelium-dependent vasodilator, or pinacidil, an ATP-sensitive K+ channel opener. Vasodilations were evaluated at the stimulation site (local) and at 500, 1,000, and 2,000 microm upstream. The vasodilator response evoked by direct arteriolar hyperpolarization induced by pinacidil decayed rapidly with distance, as expected for the passive spread of an electrical signal. Deletion of the gap junction proteins connexin37 or connexin40 did not alter the conduction of pinacidil-induced vasodilation. In contrast to pinacidil, the vasodilator response activated by ACh spread along the entire vessel without decrement. Although the ACh-induced conducted vasodilation was similar in wild-type and connexin37 knockout mice, deletion of connexin40 converted the nondecremental conducted response activated by ACh into one similar to that of pinacidil, with a decline in magnitude along the vessel length. These results suggest that ACh activates a mechanism of regenerative conduction of vasodilator responses. Connexin40 is essential for the ACh-activated regenerative vasodilator mechanism. However, neither connexin40 nor connexin37 is indispensable for the electrotonic spread of hyperpolarizing signals.

Fig. 1.

Time course of local and conducted vasodilation induced by pinacidil in wild-type mice. A short segment of the cremasteric arteriole was stimulated with a pressure-pulse ejection via micropipette of the ATP-sensitive K⁺ (K_ATP) channel opener pinacidil, and the resultant vasodilator responses were observed at the stimulation pipette site (local) and at locations 500, 1,000, and 2,000 μm upstream. Note that the changes in diameter decayed rapidly along the vessel length. Arrows indicate the time at which the stimulus was applied.

Fig. 2.

Analysis of the maximal response induced by pinacidil in wild-type (WT), connexin40 (Cx40) knockout (Cx40^−/−), and connexin37 (Cx37) knockout (Cx37^−/−) mice. A short segment of the arteriole was stimulated with a pulse of the K_ATP opener pinacidil ejected by pressure from a micropipette. The maximal vasodilator response induced by pinacidil was evaluated at the stimulation pipette site (local) and at locations 500, 1,000, and 2,000 μm upstream.

Fig. 3.

Time course of the local and conducted vasodilation induced by ACh in WT, Cx37^−/−, and Cx40^−/− mice. ACh was ejected by a pressure pulse (300 ms) via a micropipette to stimulate a short segment of the cremasteric arteriole, and the vasodilator response was analyzed at the stimulation site (local) and at locations at 500, 1,000, and 2,000 μm upstream. The vasodilator responses initiated by ACh in two different groups of experiments performed in WT animals were compared with the vasodilation observed in arterioles from Cx37^−/− (A) and Cx40^−/− (B) animals. Arrows indicate the time at which the stimulus was applied. *P < 0.05 vs. WT mice by one-way ANOVA plus the Newman-Keuls post hoc test.

Fig. 4.

Comparison of the peak response induced by pinacidil with that initiated by ACh in WT and Cx40^−/− mice. Maximal vasodilator responses of the data shown in Figs. 1 and 3 were compared. For this analysis, all the ACh-induced vasodilator responses shown in Fig. 3, A and B, were pooled together. *P < 0.05 vs. ACh-evoked vasodilation in WT mice by one-way ANOVA plus the Newman-Keuls post hoc test.

Fig. 5.

Time course of the conduction of maximal vasodilation induced by ACh in WT and Cx40^−/− mice. ACh was applied as described in Fig. 3, but the duration of the pressure pulse was increased to 700 ms to stimulate maximal vasodilation in the segment of the arteriole just underneath the micropipette. The vasodilator response was observed at the stimulation site (local) and at locations at 500, 1,000, and 2,000 μm upstream. Arrows indicate the time at which the stimulus was applied. *P < 0.05 vs. WT mice by one-way ANOVA plus the Newman-Keuls post hoc test.

Fig. 6.

Systolic blood pressure (SBP) of WT, Cx37^−/−, and Cx40^−/− mice. Deletion of Cx37 did not alter the arterial blood pressure, but elimination of Cx40 resulted in marked hypertension. Numbers inside the bars indicate n values. *P < 0.05 vs. WT or Cx37^−/− mice by one-way ANOVA plus the Newman-Keuls post hoc test.

Fig. 7.

Cellular distribution of Cx37 and Cx40 in the arteriolar wall. Cx40 (top) and Cx37 (bottom) were detected in endothelial cells of the arterioles of WT mice (left). There was no immunoreactivity for Cx40 or Cx37 in arterioles of Cx40^−/− and Cx37^−/− animals, respectively (right). The fluorescent signal was not evident in smooth muscle cells of WT or knockout mice. Arrows indicate the internal elastic lamina (IEL).