Evidence for signaling via gap junctions from smooth muscle to endothelial cells in rat mesenteric arteries: possible implication of a second messenger.

We investigated heterocellular communication in rat mesenteric arterial strips at the cellular level using confocal microscopy. To visualize Ca(2+) changes in different cell populations, smooth muscle cells (SMCs) were loaded with Fluo-4 and endothelial cells (ECs) with Fura red. SMC contraction was stimulated using high K(+) solution and Phenylephrine. Depending on vasoconstrictor concentration, intracellular Ca(2+) concentration ([Ca(2+)](i)) increased in a subpopulation of ECs 5-11s after a [Ca(2+)](i) rise was observed in adjacent SMCs. This time interval suggests chemical coupling between SMCs and ECs via gap junctions. As potential chemical mediators we investigated Ca(2+) or inositol 1,4,5-trisphosphate (IP(3)). First, phospholipase C inhibitor U-73122 was added to prevent IP(3) production in response to the [Ca(2+)](i) increase in SMCs. In high K(+) solution, all SMCs presented global and synchronous [Ca(2+)](i) increase, but no [Ca(2+)](i) variations were detected in ECs. Second, 2-aminoethoxydiphenylborate, an inhibitor of IP(3)-induced Ca(2+) release, reduced the number of flashing ECs by 75+/-3% (n = 6). The number of flashing ECs was similarly reduced by adding the gap junction uncoupler palmitoleic acid. Thus, our results suggest a heterocellular communication through gap junctions from SMCs to ECs by diffusion, probably of IP(3).

Ca2+ dynamics in a population of smooth muscle cells: modeling the recruitment and synchronization.

Many experimental studies have shown that arterial smooth muscle cells respond with cytosolic calcium rises to vasoconstrictor stimulation. A low vasoconstrictor concentration gives rise to asynchronous spikes in the calcium concentration in a few cells (asynchronous flashing). With a greater vasoconstrictor concentration, the number of smooth muscle cells responding in this way increases (recruitment) and calcium oscillations may appear. These oscillations may eventually synchronize and generate arterial contraction and vasomotion. We show that these phenomena of recruitment and synchronization naturally emerge from a model of a population of smooth muscle cells coupled through their gap junctions. The effects of electrical, calcium, and inositol 1,4,5-trisphosphate coupling are studied. A weak calcium coupling is crucial to obtain a synchronization of calcium oscillations and the minimal required calcium permeability is deduced. Moreover, we note that an electrical coupling can generate oscillations, but also has a desynchronizing effect. Inositol 1,4,5-trisphosphate diffusion does not play an important role to achieve synchronization. Our model is validated by published in vitro experiments obtained on rat mesenteric arterial segments.

Calcium dynamics and vasomotion in rat mesenteric arteries.

Smooth muscle cell calcium dynamics and diameter were measured in intact pressurized rat mesenteric artery segments during vasoconstriction and vasomotion. Arteries showed a certain norepinephrine (NE) threshold (0.3-0.4 microM) for the onset of vasomotion, during a cumulative NE concentration-response curve. This was due to a necessary [Ca2+]i threshold (increase over basal level of 22.2 +/- 2.6%) to elicit oscillations. The calcium oscillations obtained were synchronous over the entire vessel length and phase-shifted (in advance by 1.7 +/- 0.3 seconds) with respect to the diameter oscillations. A similar result was obtained using a KCl depolarization to contract the arteries, even though the [Ca2+]i threshold was much smaller in this case (increase over basal level of 9.9 +/- 4.3%), as compared with the NE-elicited vasomotion. Blockade of the Na+/K+-ATPase with 1 microM ouabain, or of the Na+/Ca2+ exchanger (NCX) with 1 microM KB-R 7943, did not abolish the calcium oscillations, thus showing that these two pumps are only modulatory elements, while on the other hand, voltage-gated calcium channels have been found to be important in the vasomotion mechanism.

Recruitment of smooth muscle cells and arterial vasomotion.

Investigating the recruitment and synchronization of smooth muscle cells (SMCs) is the key to understanding the physical mechanisms leading to contraction and spontaneous diameter oscillations of arteries, called vasomotion. We improved a method that allows the correlation of calcium oscillations (flashing) of individual SMCs with mean calcium variations and arterial contraction using confocal microscopy. Endothelium-stripped rat mesenteric arteries were cut open, loaded with dual calcium fluorescence probes, and stimulated by increasing concentrations of the vasoconstrictors phenylephrine (PE) and KCl. We found that the number and synchronization of flashing cells depends on vasoconstrictor concentration. At low vasoconstrictor concentration, few cells flash asynchronously and no local contraction is detected. At medium concentration, recruitment of cells is complete and synchronous, leading to strip contraction after KCl stimulation and to vasomotion after PE stimulation. High concentration of PE leads to synchronous calcium oscillations and fully contracted vessels, whereas high concentration of KCl leads to a sustained nonoscillating increase of calcium and to fully contracted vessels. We conclude that the number of simultaneously recruited cells is an important factor in controlling rat mesenteric artery contraction and vasomotion.

Role of membrane potential in vasomotion of isolated pressurized rat arteries.

Vasomotion, the phenomenon of vessel diameter oscillation, regulates blood flow and resistance. The main parameters implicated in vasomotion are particularly the membrane potential and the cytosolic free calcium in smooth muscle cells. In this study, these parameters were measured in rat perfused-pressurized mesenteric artery segments. The application of norepinephrine (NE) caused rhythmic diameter contractions and membrane potential oscillations (amplitude; 5.3 +/- 0.3 mV, frequency; 0.09 +/- 0.01 Hz). Verapamil (1 microM) abolished this vasomotion. During vasomotion, 10(-5) M ouabain (Na(+)-K(+) ATPase inhibitor) decreased the amplitude of the electrical oscillations but not their frequency (amplitude; 3.7 +/- 0.3 mV, frequency; 0.08 +/- 0.002 Hz). Although a high concentration of ouabain (10(-3) M) (which exhibits non-specific effects) abolished both electrical membrane potential oscillations and vasomotion, we conclude that the Na+-K+ ATPase could not be implicated in the generation of the membrane potential oscillations. We conclude that in rat perfused-pressurized mesenteric artery, the slow wave membrane type of potential oscillation by rhythmically gating voltage-dependent calcium channels, is responsible for the oscillation of intracellular calcium and thus vasomotion.