Cannabinoid receptor type 1 (CBR) inhibits hypothalamic leptin signaling via β-arrestin1 in complex with TC-PTP and STAT3.

Molecular interactions between anorexigenic leptin and orexigenic endocannabinoids, although of great metabolic significance, are not well understood. We report here that hypothalamic STAT3 signaling in mice, initiated by physiological elevations of leptin, is diminished by agonists of the cannabinoid receptor 1 (CBR). Measurement of STAT3 activation by semi-automated confocal microscopy in cultured neurons revealed that this CBR-mediated inhibition requires both T cell protein tyrosine phosphatase (TC-PTP) and β-arrestin1 but is independent of changes in cAMP.

Optimization of the Heterologous Expression of the Cannabinoid Type-1 (CB) Receptor.

The G protein-coupled type 1 cannabinoid receptor (CBR) mediates virtually all classic cannabinoid effects, and both its agonists and antagonists hold major therapeutic potential. Heterologous expression of receptors is vital for pharmacological research, however, overexpression of these proteins may fundamentally alter their localization pattern, change the signalling partner preference and may also spark artificial clustering. Additionally, recombinant CBRs are prone to intense proteasomal degradation, which may necessitate substantial modifications, such as N-terminal truncation or signal sequence insertion, for acceptable cell surface expression.

Mitochondrial cAMP exerts positive feedback on mitochondrial Ca uptake via the recruitment of Epac1.

We have previously demonstrated in H295R adrenocortical cells that the Ca-dependent production of mitochondrial cAMP (mt-cAMP) by the matrix soluble adenylyl cyclase (sAC; encoded by ) is associated with enhanced aldosterone production. Here, we examined whether mitochondrial sAC and mt-cAMP fine tune mitochondrial Ca metabolism to support steroidogenesis. Reduction of mt-cAMP formation resulted in decelerated mitochondrial Ca accumulation in intact cells during K-induced Ca signalling and also in permeabilized cells exposed to elevated perimitochondrial [Ca].

Calcium-dependent mitochondrial cAMP production enhances aldosterone secretion.

Glomerulosa cells secrete aldosterone in response to agonists coupled to Ca(2+) increases such as angiotensin II and corticotrophin, coupled to a cAMP dependent pathway. A recently recognized interaction between Ca(2+) and cAMP is the Ca(2+)-induced cAMP formation in the mitochondrial matrix. Here we describe that soluble adenylyl cyclase (sAC) is expressed in H295R adrenocortical cells.

Mitochondrial Ca2+ uptake correlates with the severity of the symptoms in autosomal dominant optic atrophy.

The most frequent form of hereditary blindness, autosomal dominant optic atrophy (ADOA), is caused by the mutation of the mitochondrial protein Opa1 and the ensuing degeneration of retinal ganglion cells. Previously we found that knockdown of OPA1 enhanced mitochondrial Ca(2+) uptake (Fülöp et al., 2011).

Extramitochondrial OPA1 and adrenocortical function.

We have previously described that silencing of the mitochondrial protein OPA1 enhances mitochondrial Ca(2+) signaling and aldosterone production in H295R adrenocortical cells. Since extramitochondrial OPA1 (emOPA1) was reported to facilitate cAMP-induced lipolysis, we hypothesized that emOPA1, via the enhanced hydrolysis of cholesterol esters, augments aldosterone production in H295R cells. A few OPA1 immunopositive spots were detected in ∼40% of the cells.

Control mechanisms of mitochondrial Ca(2+) uptake - feed-forward modulation of aldosterone secretion.

Mitochondrial Ca(2+) signal activates metabolism by boosting pyridine nucleotide reduction and ATP synthesis or, if Ca(2+) sequestration is supraphysiological, may even lead to apoptosis. Although the molecular background of mitochondrial Ca(2+) uptake has recently been elucidated, the regulation of Ca(2+) handling is still not properly clarified. In human adrenocortical H295R cells we found a regulatory mechanism involving p38 MAPK and novel-type PKC isoforms.

Molecular and functional characterization of Hv1 proton channel in human granulocytes.

Voltage-gated proton current (I(Hv)) has been characterized in several cell types, but the majority of the data was collected in phagocytes, especially in human granulocytes. The prevailing view about the role of I(Hv) in phagocytes is that it is an essential supporter of the intense and sustained activity of Nox2 (the core enzyme of the phagocyte NADPH oxidase complex) during respiratory burst. Recently H(v)1, a voltage-gated proton channel, was cloned, and leukocytes from H(v)1 knockout mice display impaired respiratory burst.

Mitochondrial Ca2+ uptake is inhibited by a concerted action of p38 MAPK and protein kinase D.

Angiotensin II elicits cytosolic Ca2+ signal that is transferred into the mitochondria. Previously we found in H295R cells that this signal transfer is enhanced by both the inhibition of p38 MAPK and a novel isoform of PKC [G. Szanda, P.

Effect of cytosolic Mg2+ on mitochondrial Ca2+ signaling.

Cytosolic Ca2+ signals are followed by mitochondrial Ca2+ uptake, which, in turn, modifies several biological processes. Mg2+ is known to inhibit Ca2+ uptake by isolated mitochondria, but its significance in intact cells has not been elucidated. In HEK293T cells, activation of purinergic receptors with extracellular ATP caused cytosolic Ca2+ signals associated with parallel changes in cytosolic [Mg2+].

Participation of p38 MAPK and a novel-type protein kinase C in the control of mitochondrial Ca2+ uptake.

Angiotensin II elicits cytosolic and mitochondrial Ca2+ signal in H295R adrenocortical cells. We found that Ca2+ uptake rate and peak values in small mitochondrial regions both depend on the colocalization of these mitochondrial regions with GFP-marked endoplasmic reticular (ER) vesicles. The dependence of the Ca2+ response on this colocalization is abolished by SB202190 and PD169316, inhibitors of p38 MAPK, as well as by transfection with siRNA against p38 MAPK mRNA.

Reactive oxygen species, Ca2+ signaling and mitochondrial NAD(P)H level in adrenal glomerulosa cells.

The acute effects of ultraviolet light, the superoxide-generating xanthine-xanthine oxidase system and H(2)O(2) to on calcium signaling and mitochondrial pyridine nucleotide metabolism were investigated in rat glomerulosa cells. UV light induced the formation of superoxide, that, similar to exogenously applied superoxide and H(2)O(2), decreased the level of mitochondrial NAD(P)H. Free radical scavengers antagonized this effect of UV light.