[Glu27]-PKC (19-36)

Identified type A photoreceptors of Hermissenda express differential effects of classical conditioning. Lateral type A photoreceptors exhibit an increase in excitability to both the conditioned stimulus (CS; light) and extrinsic current. In contrast, medial type A photoreceptors do not express enhanced excitability, but do show enhancement of the medial B to medial A synaptic connection. Therefore, both enhanced excitability and changes in synaptic strength may contribute to long-term plasticity underlying classical conditioning. The activation of protein kinase C (PKC) is involved in the induction of enhanced excitability of identified type B photoreceptors produced by one-trial conditioning and the expression of enhanced excitability in B photoreceptors after multitrial classical conditioning. We have examined a possible role for persistent kinase activity in the expression of enhanced excitability in lateral type A photoreceptors and enhancement of the medial B to medial type A synaptic connection after classical conditioning. Injection of the PKC inhibitor peptide PKC(19-36) into medial type B photoreceptors of conditioned animals did not significantly change the amplitude of medial A IPSPs elicited by single spikes in the medial B photoreceptor. Injections of PKC(19-36) into medial B photoreceptors of pseudorandom controls also did not significantly change the amplitude of IPSPs recorded from the medial A photoreceptor. In contrast, spikes elicited by extrinsic current in lateral type A photoreceptors of conditioned animals were significantly reduced in frequency after intracellular injection of PKC(19-36) as compared with pseudorandom controls. Injection of the noninhibitory analog peptide [glu27]PKC(19-36) did not affect excitability. Thus, enhanced excitability in the lateral A photoreceptor of conditioned animals seems to be influenced, in part, by a constitutively active kinase or a persistent kinase activator, whereas synaptic enhancement of the connection between the medial B and medial A photoreceptors of conditioned animals may involve a different mechanism.

The role of calcium in the action of tumor necrosis factor (TNF) on human neutrophils is not clear. With fluorescent cytometry, using the visible wavelength calcium probe, fluo-3, and patch clamping, we investigated whether TNF induces cytosolic free Ca2+ changes and Ca(2+)-activated Cl- current, respectively. Bath application of 1000 units/ml recombinant human TNF alpha (rhTNF alpha) induced a rise in cytosolic free Ca2+ in 75% of fluo-3-loaded cells, 25% of which displayed irregular patterns of oscillation. Addition of rhTNF alpha activated Cl- current in 80% of tested cells; the activated current was blocked by 10 microM 5-nitro-2-3-phenylpropylamino)benzoic acid, a Cl- channel blocker. The current was similarly activated by 1 microM ionomycin, a Ca2+ ionophore. To study the mechanism by which rhTNF alpha induced Ca(2+)-activated Cl- current, we examined the involvement of calcium/calmodulin-dependent protein kinase (CaM kinase). With intracellular application of the Ca2+ chelator 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (5 mM), the calmodulin antagonist (2 microM), CaM kinase II-(290-309), or the inhibitory peptide (10 microM), CaM kinase II-(273-302), the current was no longer activated by rhTNF alpha. The intracellular application of the control peptide (10 microM), CaM kinase II-(284-302), or the protein kinase C (PKC) inhibitory, PKC-(19-36), or control, [Glu27]PKC-(19-36), peptide (5 microM) did not block the rhTNF alpha-induced Cl- current. These results show that Ca2+ changes are associated with the effects of rhTNF alpha and that CaM kinase plays a role in the mechanism underlying rhTNF alpha-induced activation of Ca(2+)-activated Cl- current in human neutrophils.

Calcium-activated, voltage-independent Cl- currents have been demonstrated in human neutrophils (Krause, K.-H., and Welsh, M.J. (1990) J. Clin. Invest. 85, 491-498). The activation is mediated by calcium/calmodulin-dependent protein kinase and not by protein kinase C (PKC) (Schumann, M., Gardner, A.P., and Raffin, T.A. (1993) J. Biol. Chem. 268, 2134-2140). It is not known whether there are Ca(2+)-independent, voltage-dependent Cl- currents in these cells. Using K(+)-free solutions and patch-clamp recordings from human neutrophils, we separated the whole cell Cl- current. Base-line Cl- currents of unstimulated cells were small and displayed time and voltage independence; some showed voltage dependence. With a Ca(2+)-free pipette solution, bath-administered 1 microM phorbol 12-myristate 13-acetate (PMA) or 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (fMLP) for 10 s induced augmented Cl- currents with voltage- and time-dependent outwardly rectifying properties. The threshold voltage of tail Cl- current activation was -69 mV. With a pipette solution containing 0.1 mM Ca2+, bath-administered 0.1 microM fMLP, 1 microM PMA, or 1 microM Ca2+ ionophore A23187 for 30 s induced augmented Cl- currents with voltage-independent properties. With intracellular application of 5 microM PKC inhibitor PKC(19-36), voltage-dependent Cl- currents were no longer activated by PMA or fMLP. Similar application of 5 microM PKC noninhibitory analog [Glu27]PKC(19-36) did not block PMA (or fMLP)-induced Cl- currents. These results indicate that, in addition to Ca(2+)-activated Cl- currents, human neutrophils have voltage-dependent Cl- currents which are regulated by PKC.