Intracellular Ca2+ and renin gene transcription

The ‘calcium paradox’ of renin suppression by elevated intracellular Ca2+ ([Ca2+]i) has long puzzled investigators concerned with the issue of coupling of intracellular signals to effector responses [1]. In most cell types, a rise of [Ca2+]i, independent of its source, results in activation of multiple responses, including secretion or release of stored bioactive compounds. The most notable exceptions to this common behaviour are represented by renin-producing juxtaglomerular cells and parathyroid cells. Again, at odds with a variety of secretory systems, juxtaglomerular cells respond to a rise of another intracellular ‘second messenger', adenosine (3′,5′)-cyclic monophosphate (cAMP), with an increase in renin release. Another related signalling nucleotide, guanosine (3′,5′)-cyclic monophosphate (cGMP), similarly coupled to various vasodilator receptors, mimicks instead the effects of [Ca2+]i, inhibiting renin release [1–3]. Both cAMP and cGMP are inhibitors of contraction, proliferation, protein synthesis and secretion in most cell systems. Therefore, it is more appropriate to speak of a ‘renin paradox’ or a ‘juxtaglomerular cell paradox', as most intracellular signals participating in renin release appear differently and uniquely coupled in this cell population, as well as in related cell types endowed with renin synthesis, storage and release capabilities. As an example, glomerular mesangial cells that share anatomical connection and a specialized smooth muscle cell phenotype with granular cells of the juxtaglomerular apparatus do show a similar intracellular signal/renin release linkage [2]. The issue is not solely relevant to the intracellular microenvironment because it is now clear that the renin-stimulating potential of certain antihypertensive drugs, such as Ca2+-channel blockers, is mediated by relief of the inhibitory effect of [Ca2+]i [3,4]. Reducing Ca2+ influx in juxtaglomerular cells results in enhancement of renin synthesis, contributing to the stimulation of the angiotensin II/aldosterone system found in patients taking this class of drugs [4]. Conversely, beta-blockers depress renin release by inhibiting the generation of intracellular cAMP, while nitrates acting through cGMP in turn stimulate renin, a response shared by a host of vasodilator drugs [3–6]. Physicians should always take into account the potential effects of antihypertensive agents on the renin–angiotensin–aldosterone (RAA) axis, particularly when other stimuli of renin, such as diuretics, are also prescribed as an association therapy. The work by Fuchs et al. [7] in this issue of the journal is an interesting investigation into the molecular mechanisms underlying this unique cellular response. Basically an extension of earlier work on parathyroid cells, the study is accurate and based on state-of-the art methodology, focusing on the role of the intracellular oxidoreductive enzyme AP endonuclease/Redox factor-1 (Ref-1) in this cellular response. This transcription factor activator, also referred to as APEX, HAP-1 and APE1, has important roles in DNA repair, antagonism of apoptosis and antioxidant responses in a variety of tissues [8–10]. An almost ubiquitous protein, Ref-1 has a molecular weight of 36.5 kDa and is encoded by a 3-kb gene located on chromosome 14. Activation of transcription factors, which occurs via a redox-based mechanism, can be tracked to an approximately 6-kDa N-terminal fragment, with Cys-65 critical to this function. The central assumption of Fuchs et al. [7] is that a negative Ca2+-responsive element (nCaRE), identical to a region of the PTH promoter, might block renin gene expression by translocating to the nucleus and binding Ref-1 following a rise of [Ca2+]i. To prove this hypothesis, the investigators first identified a nCaRE sequence 1800 bp upstream of the transcription start site of the human renin proximal promoter in renin-expressing chorio-decidual cells. Subsequent elegant transient expression experiments with three different constructs lacking or incorporating the key sequence of nCaRE demonstrated that varying [Ca2+]i itself is not sufficient to block renin gene expression, if a proper negative response element is absent. Mutation of the palindromic sequence of nCaRE expectedly decreased transcriptional activity. The data are consistent with a genomic control for renin biosynthesis, although potential effects of [Ca2+]i on the release of previously stored prorenin/renin should also be considered. In evaluating these results, the time course of the renin responses/nuclear translocation events should always be kept in mind, as the [Ca2+]i movements are physiologically short-lived, while genomic responses take minutes to hours to occur. It is often difficult to reconcile immediate, ‘flash’ signals such as the [Ca2+]i bursts in response to hormones or vasoactive agents, with long-term regulatory responses, as in the case of renin synthesis. Nevertheless, because the RAA locally reacts to changes in glomerular perfusion pressure, a logical basis for rapid adjustments of renin synthesis can easily be envisioned. A further step was to link the nuclear protein Ref-1 to the nCaRE sequence, a task accomplished by electrophoresis mobility shift assays, in which a Ref-1 antibody supershifted one of the nucleoprotein complexes obtained. Ref-1 migrated from the cytoplasm to the nucleus within 2 h of a rise of [Ca2+]i, either triggered by a ionophore or by angiotensin II. A topic of relevance is that despite substantial identity with transcription factors and Ca2+-responsive elements of parathyroid cells, the renin-producing cells employed herein lack the Ca2+-sensing receptor [11]. This cation sensor has been thoroughly exploited in studying parathyroid cells, osteoclasts and renal tubular cells of various origin, resulting in a major advance in the understanding of how extracellular Ca2+ levels affect the function of cells implicated in Ca2+ homeostasis [11,12]. The Ca2+-sensing receptor relies upon a phospholipase C-coupled signalling sequence involving a rise of [Ca2+]i, mostly through discharge of intracellular stores [11,12]. Its absence implies that juxtaglomerular cells do not monitor ambient Ca2+, but rather respond to fluxes across the plasma membrane, providing a clue as to the renin-stimulating effects of voltage-gated Ca2+-channel blockers. However, the study by Fuchs et al. [7] does not clarify which mechanism(s) of [Ca2+]i elevation might be directly involved in renin suppression. Several candidates should be considered, including the release of stored Ca2+ and the subsequent operation of store-gated Ca2+ influx channels, parallel or alternative to VOC [13,14]. Indeed, the Ca2+ ionophore ionomycin employed by Fuchs et al. does not help to discriminate the source of increased Ca2+, as it permeabilizes all cellular membranes equilibrating intra- and extracellular [Ca2+], including Ca2+ stores in the endoplasmic reticulum cisternae. The use of angiotensin II appears more relevant to physiologic regulation of renin secretion, as the peptide is obviously implicated in the feedback control of renin, and has access to cellular Ca2+ pools and channels through receptor-operated mechanisms. With respect to this issue, better targeted protocols employing selective Ca2+ probes should be used. Ryanodine, thapsigargin, gadolinium and a host of divalent cations such as Cd2+, Zn2+, Mn2+ are only a brief list of effective tools that could be employed in future work to dissect out the various cellular Ca2+ currents that may have an impact on the nCaRE regulatory sequence [13,14]. Obviously, Ca2+-channel blockers may also prove useful in this context. A more accurate picture of the molecular pathways that control renin synthesis and release, as it may stem out of studies such as the one by Fuchs et al. [7], would be a welcome development in a field of vascular biology with remarkable clinical and pharmacological implications.