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It has been suggested that L-type [Ca.sup.2+] channels play an important role in cell swelling-induced vasoconstriction. However, there is no direct evidence that [Ca.sup.2+] channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type [Ca.sup.2+] channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein kinase activation. [Ba.sup.2+] currents ([I.sub.Ba]) through L-type [Ca.sup.2+] channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein with the conventional whole cell patch-clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced [Ca.sup.2+] channel activity but did not alter the voltage-dependent characteristics of [Ca.sup.2+] channels. Bath application of selective inhibitors of protein kinase C (PKC), Ro-31-8425 or Go-6983, prevented [I.sub.Ba] enhancement by hypotonic swelling, whereas the specific protein kinase A (PKA) inhibitor KT-5720 had no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased [I.sub.Ba], under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on [I.sub.Ba] when cells were first exposed to hypotonic solution. Furthermore, downregulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance [Ca.sup.2+] channel activity in rabbit portal vein smooth muscle cells through activation of PKC. cell swelling; protein kinases; calcium current

Experiments were performed to study the regions of the octapeptide angiotensin II (ANG II) type I (AT1) receptor involved in the initiation of intracellular changes that lead to changes in intracellular K (IK) and intracellular Ca (ICa). Results indicated the importance of the third cytoplasmic loop of the AT1a receptor in the activation of G protein and the regulation of IK and ICa. A mechanism that was postulated for the significance of the third loop in the processes was its ability to bind to ANG II and change the conformation of the G protein.

An investigation of voltage-dependent potassium and calcium conductance elicited by angiotensin II (ANG II) in neurons isolated from Sprague-Dawley rats showed that protein kinase C (PKC) and inositol 1,4,5-triphosphate (IP3) play an important role in the modulation of these ion currents after ANG II type 1 receptor activation. Addition of PKC antagonists further enhanced the ANG II-induced decrease in K+ current and abolished the ANG II-induced increase in Ca2+ current, whereas addition of IP3 reduced K+ current.

Analysis of receptor-G protein coupling processes in rat brain stem and hypothalamus by injecting a 22-amino acid peptide (PEP-22) that corresponds to the third intracellular loop of cloned AT2 receptor shows that PEP-22 induces an increase in rectifier K+ current in the neurons, this effect being similar to effect of angiotensin II (ANG II). Superfusion of PP2A inhibitor okadaic acid and treatment with pertussis toxin suppress the effect of PEP-22, indicating that PP2A and Gi proteins contribute to the PEP-22-induced effect, respectively. The third intracellular loop of AT2 receptor is vital for ANG II-induced activation of neuronal K+ current.

Pretreatment of cultures with pertussis toxin and intracellular administering of an antibody against the inhibitory guanine nucleotide (GTP) binding protein nullify AT2-receptor-mediated stimulation of neuronal IK by angiotensin II (ANG II). However, AT2-receptor-mediated stimulation remained unaffected when antibodies of other GTP binding proteins were used. The serine/threonine phosphatase inhibitor okadaic acid and anti-type 2A protein phosphatase antibodies can also control the effect of ANG II, unlike the tyrosine phosphatase inhibitor sodium orthovanadate.

Discrete AT2 receptor-mediated effects of angiotensin II (ANG II) were proved in IA and IK in neonate neurons that were specially cultured. Data obtained from the study provided an electrophysiological basis for behavioral and physiological effects induced by the ANG II receptor subtype within the brain. Results of previous studies suggested that ANG II induces an augmentation in the net outward ionic current within neurons that are cocultured from neonate rat hypothalamus and brain stem. In the present study, whole cell voltage clamp methods were employed to determine the currents regulated by AT2 receptors. It was concluded that transient K+ current and delayed-rectifier K+ current are induced in cultured neurons.

It has been suggested that L-type Ca2+ channels play an important role in cell swelling-induced vasoconstriction. However, there is no direct evidence that Ca2+ channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca2+ channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein kinase activation. Ba2+ currents (1Ba) through L-type Ca2+ channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein with the conventional whole cell patch-clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca2+ channel activity but did not alter the voltage-dependent characteristics of Ca2+ channels. Bath application of selective inhibitors of protein kinase C (PKC), Ro-31-8425 or Go-6983, prevented lBa enhancement by hypotonic swelling, whereas the specific protein kinase A (PKA) inhibitor KT-5720 had no effect. Bath application of phorbol 12,13 dibutyrate (PDBu) significantly increased 1Ba under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on 1Ba when cells were first exposed to hypotonic solution. Furthermore, downregulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance Ca2+ channel activity in rabbit portal vein smooth muscle cells through activation of PKC. [ABSTRACT FROM AUTHOR]