## Open Access Dissertations and Theses

1993

Thesis

#### Degree Name

Doctor of Philosophy (PhD)

#### Department

Biomedical Sciences

E.E. Daniel

#### Abstract

Endothelial cells contain a high density of vasoactive intestinal peptide (VIP) receptors. Since VIP is a potent vasodilator, activation of these receptors may lead to the release of endothelium-dependent relaxing factor (EDRF). Using patch-clamp techniques it was demonstrated that VIP modulates plasma membrane K$\sp+$ channels in bovine pulmonary artery endothelial cells. VIP inhibited activity of inwardly rectifying K$\sp+$ channels (I$\sb{\rm Kin}$) and activated opening of the Ca$\sp{2+}$-dependent K$\sp+$ channels (K$\sb{\rm Ca}$). Activation of K$\sb{\rm Ca}$ channels tends to hyperpolarize the cell membrane. This transient hyperpolarization increases the driving force for influx of extracellular Ca$\sp{2+}$ through nonselective cation channels. This way VIP may contribute to Ca$\sp{2+}$ influx necessary for the potential EDRF production.

Although VIP activates adenylate cyclase and production of cyclic AMP (cAMP) in many cell types, this study provides evidence allowing us to exclude cAMP as a second messenger for VIP action in endothelial cells. Direct evidence comes from the measurements of cAMP level in bovine pulmonary artery endothelial cells stimulated with VIP and isoproterenol. Isoproterenol increased cAMP level, whereas VIP did not, or even decreased cAMP production in some cases. Indirect evidence comes from the results of patch-clamp experiments. VIP strongly suppressed I$\sb{\rm Kin}$ channel activity, whereas the inhibitory effect of isoproterenol on this channel was very weak (about 20% of the VIP effect). cAMP elevation may reduce the activity of I$\sb{\rm Kin}$ channel, but is not a mediator for the inhibitory effect of VIP on these channels. In addition, VIP was still able to inhibit I$\sb{\rm Kin}$ channel activity in outside-out patches, where the second messenger system is not operating. This effect was mediated by a G protein that most likely couples directly to the channel. This unidentified G protein revealed GTP-$\gamma$-S and cholera toxin sensitivity and resistance to pertussis toxin.

Since elevated intracellular Ca$\sp{2+}$ is a trigger for EDRF production, it has been demonstrated that modulation of the calcium fluxes at the level of internal stores did influence an influx of extracellular Ca$\sp{2+}$. Cyclopiazonic acid (CPA), an inhibitor of SR/ER Ca$\sp{2+}$ pump, (i) induced K$\sb{\rm Ca}$ currents, presumably as a consequence of the spontaneous leakage of Ca$\sp{2+}$ from internal stores, (ii) reduced the I$\sb{\rm Kin}$ currents, and (iii) enhanced influx of extracellular Ca$\sp{2+}$ through nonselective cation channels. Moreover, CPA activated K$\sb{\rm Ca}$ currents in endothelial cells by a mechanism independent of 1,4,5-triphosphate (IP$\sb3$). An expected consequence of this action of CPA may be an influx of extracellular Ca$\sp{2+}$. In parallel studies, it has been demonstrated that CPA relaxed rat aorta in endothelium-dependent manner, suggesting CPA-induced release of EDRF. Ca$\sp{2+}$ enters the endothelial cell through nonselective cation channels, which can be activated by an agonist or a mechanical stimulus (e.g. stretch). It is still unclear whether these nonselective cation channels in bovine pulmonary artery endothelial cells are controlled by free intracellular Ca$\sp{2+}$, empty Ca$\sp{2+}$ ER stores or an agonist.

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