Protein kinase C regulates ionic conductance in hippocampal pyramidal neurons: electrophysiological effects of phorbol esters.

Abstract

The vertebrate central nervous system contains very high concentrations of protein kinase C, a calciumand phospholipid-stimulated phosphorylating enzyme. Phorbol esters, compounds with inflammatory and tumor-promoting properties, bind to and activate this enzyme. To clarify the role of protein kinase C in neuronal function, we have localized phorbol ester receptors in the rat hippocampus by autoradiography and examined the electrophysiological effects of phorbol esters on hippocampal pyramidal neurons in vitro. Phorbol esters blocked a calcium-dependent potassium conductance. In addition, phorbol esters blocked the late hyperpolarization elicited by synaptic stimulation even though other synaptic potentials were not affected. The potencies of several phorbol esters in exerting these actions paralleled their affinities for protein kinase C, suggesting that protein kinase C regulates membrane ionic conductance.Protein kinase C, a phosphorylating enzyme stimulated by calcium and phospholipid, occurs ubiquitously in the body but is most concentrated in the brain (1-4). In peripheral tissues, protein kinase C may mediate cellular responses to membrane receptor stimulation; however, its role in the central nervous system is largely unknown (5-10). Phorbol esters, lipophilic compounds with inflammatory and tumorpromoting properties, may facilitate studies of protein kinase C, since they directly stimulate this enzyme (4, 11-13). Phorbol esters have been used as probes to demonstrate a role of protein kinase C in regulating effects of neurotransmitters on smooth muscle (14). To clarify the role of protein kinase C in neuronal function, we have now localized phorbol ester receptors in the rat hippocampus by autoradiography and studied the effects of phorbol esters on hippocampal pyramidal neurons by using intracellular recording techniques in the slice preparation in vitro. Phorbol esters potently affect neuronal electrophysiological properties, suggesting that protein kinase C regulates membrane ionic conductance. MATERIALS AND METHODSAutoradiography. Autoradiography was performed according to the methods of Palacios et al. (15) and of Nagle and Blumberg (16). Rats were perfused via the left ventricle with phosphate-buffered saline followed by 0.32 M sucrose. Eight-micrometer-thick brain sections were cut on a cryostat and thaw-mounted onto microscope slides coated with chrome alum/gelatin. Slide-mounted tissue sections were incubated at room temperature with 2-3 nM 3H-labeled phorbol 12,13-dibutyrate (PBt2) (New England Nuclear) for 45 min in buffer containing 50 mM Tris HCl at pH 7.7 and 100 mM NaCl. To determine nonspecific binding, adjacent sections were incubated in the same medium with the addition of 1 uM unlabeled PBt2. After incubation, tissue sections were washed for 4 min at 40C, dipped in deionized water, and dried rapidly under a stream of cold dry air. After this labeling procedure, the slide-mounted tissue sections were juxtaposed to a tritium-sensitive film (Ultrafilm, LKB, Bromma, Sweden) in a s...

Protein kinase C activators block specific calcium and potassium current components in isolated hippocampal neurons

“…Effects of PKC activators on K currents Bath perfusion of active phorbol esters increases both the amplitude and rate of rise of the Ca action potential recorded in CA1 pyramidal cells in the hippocampal slice (Baraban et al, 1985;Malenka et al, 1986b). Blockade of K conductances could explain the increase in spike parameters.…”

“…In hippocampus, depression of ZK.ea may be partly accounted for by depression of I,,. However, the membrane hyperpolarization caused by Z,-, is far more sensitive to blockade by phorbol esters than the Ca action potential (Baraban et al, 1985;Malenka et al, 1986b), so a direct effect on Ca-dependent K conductances is also possible. Since these currents influence neuronal excitability in a variety of ways, it seems that PKC must subserve a variety of functions in different species.…”

“…Effects produced by prolonged application of phorbol esters to tissue slices are only very slowly reversible (Baraban et al, 1985;Malenka et al, 1986a, b). This might indicate either that the effects themselves are inherently long-lived or that it is difficult to wash these lipophilic compounds from the tissue.…”

“…Phorbol esters block M-current in neuroblastoma cell lines (Higashida and Brown, 1986) but not in the hippocampus (Malenka et al, 1986b). ZK.ca, on the other hand, is suppressed by PKC activators in a number of systems (Baraban et al, 1985;Freedman and Aghajanian, 1985;Alkon et al, 1986;Farley and Auerbath, 1986;Malenka et al, 1986b). In tissue-cultured central neurons, ZK is also reduced (Grega et al, 1987), but Z, and Z,., were not analyzed in those cells.…”

“…Data from the hippocampal slice had indicated that phorbol esters blocked a Ca-dependent K conductance (Baraban et al, 1985;Malenka et al, 1986). Our results confirm this observation in acutely isolated cells.…”