Regulation of histamine release in rat hypothalamus and hippocampus by presynaptic galanin receptors.

The effect of galanin, a peptide present in a subpopulation of histaminergic neurons emanating from the rat posterior hypothalamus, was investigated on K(+)-evoked [3H]histamine release in slices and synaptosomes from rat cerebral cortex, striatum, hippocampus and hypothalamus. Porcine galanin (0.3 microM) significantly inhibited histamine release induced by 25 mM K+ in slices from hypothalamus and hippocampus, but not from cerebral cortex and striatum, i.e., only in regions in which a colocalization of histamine and galanin has been described. The inhibitory effect of galanin was concentration dependent, with an EC50 value of 5.8 +/- 1.9 nM. The maximal inhibition was of 30-40% in hypothalamic and hippocampal slices depolarized with 25 mM K+. The galanin-induced inhibition observed in hypothalamic slices was not prevented in the presence of 0.6 microM tetrodotoxin and also occurred in hippocampal and hypothalamic synaptosomes, strongly suggesting the activation by galanin of presynaptic receptors located upon histaminergic nerve endings. The maximal inhibitory effect of galanin in slices or synaptosomes was lower than that previously reported for histamine acting at H3-autoreceptors, possibly suggesting that not all histaminergic axon terminals, even in the hypothalamus and hippocampus, are endowed with galanin receptors. It increased progressively in hypothalamic and hippocampal synaptosomes as the strength of the depolarizing stimulus was reduced. It is concluded that galanin modulates histamine release via presynaptic receptors, presumably autoreceptors located upon nerve terminals of a subpopulation of cerebral histaminergic neurons.

Phencyclidine blocks histamine H3-receptors in rat brain.

Phencyclidine (PCP) was examined for its ability to modulate histamine release from rat brain slices labeled with L-[3H]histidine. PCP failed to mimic but completely reversed the autoinhibitory effect of histamine at H3-receptors with an apparent Ki value of 13 +/- 3 microM. A direct interaction of PCP with H3-autoreceptors rather than PCP or sigma receptor sites was confirmed by binding studies. PCP inhibited the binding of [3H](R)alpha-methylhistamine to H3-receptor sites in rat cerebral membranes with a Ki value of 25 +/- 2 microM. It is concluded that PCP is a H3-receptor antagonist of moderate potency.

Protein kinase C isoforms in rat kidney proximal tubule: acute effect of angiotensin II.

The present study examined the effect of phorbol esters, Ca2+, and angiotensin II (ANG II) on protein kinase C (PKC) isoforms in the rat proximal tubule. The immunoblot analysis of PKC isoforms of particulate and cytosolic fractions of proximal tubules revealed immunoreactive proteins when antibodies against PKC-alpha, -delta, -epsilon, and -zeta, but not -beta and -gamma were used. Phorbol dibutyrate (PDBU) induced the translocation of PKC-alpha, -delta, and -epsilon, whereas an inactive phorbol ester had no effect. PDBU and ionomycin increased particulate PKC specific activity from 0.67 +/- 0.09 to 1.56 +/- 0.18 and 0.96 +/- 0.04 pmol.microgram protein-1.2 min-1, respectively. ANG II (10(-7) M) induced a time-dependent increase in particulate PKC-alpha immunoreactivity observed after 2 min and maintained for 12 min. Particulate PKC-epsilon immunoreactivity increased after 4 min. Meanwhile, PKC-delta and -zeta were not modified by ANG II. Accordingly, ANG II elicited a rise in the specific activity of the particulate PKC, which increased to 0.89 +/- 0.09 pmol.micrograms protein-1.2 min-1 after 2 min. This was inhibited by a preincubation in the presence of 10(-5) M losartan, specific inhibitor of angiotensin subtype 1 receptors. These data indicate that PKC-alpha and -epsilon are potential candidates to regulate the activity of Na+/H+ and Na(+)-HCO3- transporters because they are translocated with a time course fitting with that of the reported effect of ANG II on those transporters.

NHE3 activity and trafficking depend on the state of actin organization in proximal tubule.

The present study was addressed to define the contribution of cytoskeleton elements in the kidney proximal tubule Na+/H+ exchanger 3 (NHE3) activity under basal conditions. We used luminal membrane vesicles (LMV) isolated from suspensions of rat cortical tubules pretreated with either colchicine (Colch) or cytochalasin D (Cyto D). Colch pretreatment of suspensions (200 microM for 60 min) moderately decreased LMV NHE3 activity. Cyto D pretreatment (1 microM for 60 min) elicited an increase in LMV NHE3 transport activity but did not increase Na-glucose cotransport activity. Cyto D pretreatment of suspensions did not change the apparent affinity of NHE3 for internal H+. In contrast, after Cyto D pretreatment of the suspensions, NHE3 protein abundance was increased in LMV and remained unchanged in cortical cell homogenates. The effect of Cyto D on NHE3 was further assessed with cultures of murine cortical cells. The amount of surface biotinylated NHE3 increased on Cyto D treatment, whereas NHE3 protein abundance was unchanged in cell homogenates. In conclusion, under basal conditions NHE3 activity depends on the state of actin organization possibly involved in trafficking processes between luminal membrane and intracellular compartment.

Regulation by PKC isoforms of Na(+)/H(+) exchanger in luminal membrane vesicles isolated from cortical tubules.

The present study was designed to determine the Na/H exchanger isoforms present in luminal membrane vesicles (LMV) isolated from rat kidney cortical tubule suspensions, as well as the effects of acute phorbol ester (phorbol myristate acetate, PMA) and angiotensin II (ANG II) pretreatment of suspensions on NHE activity and protein kinase C (PKC) isoform abundance. In LMV, both NHE3 and NHE2 proteins were found by Western blot analysis, but only ethylisopropylamiloride-sensitive and almost completely Hoe-694-resistant Na/H exchange activity was observed from (22)Na uptake and thus attributed to NHE3. PMA pretreatment increased Na/H exchange activity and PKC isoforms alpha, delta, and epsilon abundance in LMV, and these effects were prevented by PKC inhibition. Low-dose ANG II (10(-11) M) pretreatment increased Na/H exchange activity and only PKC-zeta abundance in LMV, and these effects were also prevented by PKC inhibition. After high-dose ANG II (10(-7) M), Na/H exchange activity was decreased in LMV. PKC inhibition did not prevent this effect. In conclusion, the stimulating effects of PMA and low-dose ANG II are explained by the translocation of different isoforms of PKC in LMV, whereas the inhibitory effect of high-dose ANG II is not PKC dependent.