Nick A. Guagliardo

TASK channel deletion in mice causes primary hyperaldosteronism

When inappropriate for salt status, the mineralocorticoid aldosterone induces cardiac and renal injury. Autonomous overproduction of aldosterone from the adrenal zona glomerulosa (ZG) is also the most frequent cause of secondary hypertension. Yet, the etiology of nontumorigenic primary hyperaldosteronism caused by bilateral idiopathic hyperaldosteronism remains unknown. Here, we show that genetic deletion of TWIK-related acid-sensitive K (TASK)-1 and TASK-3 channels removes an important background K current that results in a marked depolarization of ZG cell membrane potential. Although TASK channel deletion mice (TASK−/−) adjust urinary Na excretion and aldosterone production to match Na intake, they produce more aldosterone than control mice across the range of Na intake. Overproduction of aldosterone is not the result of enhanced activity of the renin–angiotensin system because circulating renin concentrations remain either unchanged or lower than those of control mice at each level of Na intake. In addition, TASK−/− mice fail to suppress aldosterone production in response to dietary Na loading. Autonomous aldosterone production is also demonstrated by the failure of an angiotensin type 1 receptor blocker, candesartan, to normalize aldosterone production to control levels in TASK−/− mice. Thus, TASK−/− channel knockout mice exhibit the hallmarks of primary hyperaldosteronism. Our studies establish an animal model of nontumorigenic primary hyperaldosteronism and identify TASK channels as a possible therapeutic target for primary hyperaldosteronism.

Zona glomerulosa cells of the mouse adrenal cortex are intrinsic electrical oscillators

Aldosterone, which plays a central role in the regulation of blood pressure, is produced by zona glomerulosa (ZG) cells of the adrenal gland. When dysregulated, aldosterone is pathogenic and contributes to the development and progression of cardiovascular and renal disease. Although sustained production of aldosterone requires persistent Ca2+ entry through low-voltage activated Ca2+ channels, isolated ZG cells are considered nonexcitable, with recorded membrane voltages that are too hyperpolarized to permit Ca2+ entry. Here, we show that mouse ZG cells within adrenal slices spontaneously generate membrane potential oscillations of low periodicity. This innate electrical excitability of ZG cells provides a platform for the production of a recurrent Ca2+ signal that can be controlled by Ang II and extracellular potassium, the 2 major regulators of aldosterone production. We conclude that native ZG cells are electrical oscillators, and that this behavior provides what we believe to be a new molecular explanation for the control of Ca2+ entry in these steroidogenic cells.

Adrenocortical Zonation Results from Lineage Conversion of Differentiated Zona Glomerulosa Cells

Lineage conversion of differentiated cells in response to hormonal feedback has yet to be described. To investigate this, we studied the adrenal cortex, which is composed of functionally distinct concentric layers that develop postnatally, the outer zona glomerulosa (zG) and the inner zona fasciculata (zF). These layers have separate functions, are continuously renewed in response to physiological demands, and are regulated by discrete hormonal feedback loops. Their cellular origin, lineage relationship, and renewal mechanism, however, remain poorly understood. Cell-fate mapping and gene-deletion studies using zG-specific Cre expression demonstrate that differentiated zG cells undergo lineage conversion into zF cells. In addition, zG maintenance is dependent on the master transcriptional regulator Steroidogenic Factor 1 (SF-1), and zG-specific Sf-1 deletion prevents lineage conversion. These findings demonstrate that adrenocortical zonation and regeneration result from lineage conversion and may provide a paradigm for homeostatic cellular renewal in other tissues.

TASK-3 Channel Deletion in Mice Recapitulates Low-Renin Essential Hypertension

Idiopathic primary hyperaldosteronism (IHA) and low-renin essential hypertension (LREH) are common forms of hypertension, characterized by an elevated aldosterone-renin ratio and hypersensitivity to angiotensin II. They are suggested to be 2 states within a disease spectrum that progresses from LREH to IHA as the control of aldosterone production by the renin-angiotensin system is weakened. The mechanism(s) that drives this progression remains unknown. Deletion of Twik-related acid-sensitive K+ channels (TASK) subunits, TASK-1 and TASK-3, in mice (T1T3KO) produces a model of human IHA. Here, we determine the effect of deleting only TASK-3 (T3KO) on the control of aldosterone production and blood pressure. We find that T3KO mice recapitulate key characteristics of human LREH, salt-sensitive hypertension, mild overproduction of aldosterone, decreased plasma-renin concentration with elevated aldosterone:renin ratio, hypersensitivity to endogenous and exogenous angiotensin II, and failure to suppress aldosterone production with dietary sodium loading. The relative differences in levels of aldosterone output and aldosterone:renin ratio and in autonomy of aldosterone production between T1T3KO and T3KO mice are reminiscent of differences in human hypertensive patients with LREH and IHA. Our studies establish a model of LREH and suggest that loss of TASK channel activity may be one mechanism that advances the syndrome of low renin hypertension.

Fungiform Taste Bud Degeneration in C57BL/6J Mice Following Chorda-Lingual Nerve Transection

Taste buds are dependent on innervation for normal morphology and function. Fungiform taste bud degeneration after chorda tympani nerve injury has been well documented in rats, hamsters, and gerbils. The current study examines fungiform taste bud distribution and structure in adult C57BL/6J mice from both intact taste systems and after unilateral chorda‐lingual nerve transection. Fungiform taste buds were visualized and measured with the aid of cytokeratin 8. In control mice, taste buds were smaller and more abundant on the anterior tip (<1 mm) of the tongue. By 5 days after nerve transection taste buds were smaller and fewer on the side of the tongue ipsilateral to the transection and continued to decrease in both size and number until 15 days posttransection. Degenerating fungiform taste buds were smaller due to a loss of taste bud cells rather than changes in taste bud morphology. While almost all taste buds disappeared in more posterior fungiform papillae by 15 days posttransection, the anterior tip of the tongue retained nearly half of its taste buds compared to intact mice. Surviving taste buds could not be explained by an apparent innervation from the remaining intact nerves. Contralateral effects of nerve transection were also observed; taste buds were larger due to an increase in the number of taste bud cells. These data are the first to characterize adult mouse fungiform taste buds and subsequent degeneration after unilateral nerve transection. They provide the basis for more mechanistic studies in which genetically engineered mice can be used. J. Comp. Neurol. 504:206–216, 2007.

Minireview: Aldosterone Biosynthesis: Electrically Gated for Our Protection

Aldosterone produced by adrenal zona glomerulosa (ZG) cells plays an important role in maintaining salt/water balance and, hence, blood pressure homeostasis. However, when dysregulated, aldosterone advances renal and cardiovascular disease states. Multiple steps in the steroidogenic pathway require Ca(2+), and the sustained production of aldosterone depends on maintained Ca(2+) entry into the ZG cell. Nevertheless, the recorded membrane potential of isolated ZG cells is extremely hyperpolarized, allowing the opening of only a small fraction of low-voltage-activated Ca(2+) channels of the Ca(v)3.x family, the major Ca(2+) conductance on the ZG cell membrane. As a consequence, to activate sufficient Ca(2+) channels to sustain the production of aldosterone, aldosterone secretagogs would be required to affect large decreases in membrane voltage, a requirement that is inconsistent with the exquisite sensitivity of aldosterone production in vivo to small changes (0.1 mm) in extracellular K(+). In this review, we evaluate the contribution of membrane voltage and voltage-dependent Ca(2+) channels to the control of aldosterone production and consider data highlighting the electrical excitability of the ZG cell. This intrinsic capacity of ZG cells to behave as electrical oscillators provides a platform from which to generate a recurring Ca(2+) signal that is compatible with the lengthy time course of steroidogenesis and provides an alternative model for the physiological regulation of aldosterone production that permits both amplitude and temporal modulation of the Ca(2+) signal.

Each Sensory Nerve Arising From the Geniculate Ganglion Expresses a Unique Fingerprint of Neurotrophin and Neurotrophin Receptor Genes

Neurons in the geniculate ganglion, like those in other sensory ganglia, are dependent on neurotrophins for survival. Most geniculate ganglion neurons innervate taste buds in two regions of the tongue and two regions of the palate; the rest are cutaneous nerves to the skin of the ear. We investigated the expression of four neurotrophins, nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), neurotrophin 3 (NT‐3), and NT‐4, and five neurotrophin receptors, trkA, trkB, trkC, p75, and truncated trkB (Trn‐B) in single sensory neurons of the adult rat geniculate ganglion associated with the five innervation fields. For fungiform papillae, a glass pipette containing biotinylated dextran was placed over the target papilla and the tracer was iontophoresed into the target papilla. For the other target fields, Fluoro‐Gold was microinjected. After 3 days, geniculate ganglia were harvested, sectioned, and treated histochemically (for biotinylated dextran) or immunohistochemically (for Fluoro‐Gold) to reveal the neurons containing the tracer. Single labeled neurons were harvested from the slides and subjected to RNA amplification and RT‐PCR to reveal the neurotrophin or neurotrophin receptor genes that were expressed. Neurons projecting from the geniculate ganglion to each of the five target fields had a unique expression profile of neurotrophin and neurotrophic receptor genes. Several individual neurons expressed more than one neurotrophin receptor or more than one neurotrophin gene. Although BDNF is significantly expressed in taste buds, its primary high affinity receptor, trkB, was not prominently expressed in the neurons. The results are consistent with the interpretation that at least some, perhaps most, of the trophic influence on the sensory neurons is derived from the neuronal somata, and the trophic effect is paracrine or autocrine, rather than target derived. The BDNF in the taste bud may also act in a paracrine or autocrine manner on the trkB expressed in taste buds, as shown by others.

TASK channels are not required to mount an aldosterone secretory response to metabolic acidosis in mice

The stimulation of aldosterone production by acidosis enhances proton excretion and serves to limit disturbances in systemic acid-base equilibrium. Yet, the mechanisms by which protons stimulate aldosterone production from cells of the adrenal cortex remain largely unknown. TWIK-related acid sensitive K channels (TASK) are inhibited by extracellular protons within the physiological range and have emerged as important regulators of aldosterone production in the adrenal cortex. Here we show that congenic C57BL/6J mice with genetic deletion of TASK-1 (K(2P)3.1) and TASK-3 (K(2P)9.1) channel subunits overproduce aldosterone and display an enhanced sensitivity to steroidogenic stimuli, including a more pronounced steroidogenic response to chronic NH(4)Cl loading. Thus, we conclude that TASK channels are not required for the stimulation of aldosterone production by protons but their inhibition by physiological acidosis may contribute to full expression of the steroidogenic response.

Role of voltage‐gated calcium channels in the regulation of aldosterone production from zona glomerulosa cells of the adrenal cortex

Zona glomerulosa cells (ZG) of the adrenal gland constantly integrate fluctuating ionic, hormonal and paracrine signals to control the synthesis and secretion of aldosterone. These signals modulate Ca2+ levels, which provide the critical second messenger to drive steroid hormone production. Angiotensin II is a hormone known to modulate the activity of voltage‐dependent L‐ and T‐type Ca2+ channels that are expressed on the plasma membrane of ZG cells in many species. Because the ZG cell maintains a resting membrane voltage of approximately −85 mV and has been considered electrically silent, low voltage‐activated T‐type Ca2+ channels are assumed to provide the primary Ca2+ signal that drives aldosterone production. However, this view has recently been challenged by human genetic studies identifying somatic gain‐of‐function mutations in L‐type CaV1.3 channels in aldosterone‐producing adenomas of patients with primary hyperaldosteronism. We provide a review of these assumptions and challenges, and update our understanding of the state of the ZG cell in a layer in which native cellular associations are preserved. This updated view of Ca2+ signalling in ZG cells provides a unifying mechanism that explains how transiently activating CaV3.2 channels can generate a significant and recurring Ca2+ signal, and how CaV1.3 channels may contribute to the Ca2+ signal that drives aldosterone production.

KCNK3 Variants Are Associated With Hyperaldosteronism and Hypertension.

Blood pressure (BP) is a complex trait that is the consequence of an interaction between genetic and environmental determinants. Previous studies have demonstrated increased BP in mice with global deletion of TASK-1 channels contemporaneous with diverse dysregulation of aldosterone production. In humans, genome-wide association studies in ≈100 000 individuals of European, East Asian, and South Asian ancestry identified a single nucleotide polymorphism (SNP) in KCNK3 (the gene encoding TASK-1) associated with mean arterial pressure. The current study was motivated by the hypotheses that (1) association of KCNK3 SNPs with BP and related traits extends to blacks and Hispanics, and (2) KCNK3 SNPs exhibit associations with plasma renin activity and aldosterone levels. We examined baseline BP measurements for 7840 participants from the Multi-Ethnic Study of Atherosclerosis (MESA), and aldosterone levels and plasma renin activity in a subset of 1653 MESA participants. We identified statistically significant association of the previously reported KCNK3 SNP (rs1275988) with mean arterial pressure in MESA blacks (P=0.024) and a nearby SNP (rs13394970) in MESA Hispanics (P=0.031). We discovered additional KCNK3 SNP associations with systolic BP, mean arterial pressure, and hypertension. We also identified statistically significant association of KCNK3 rs2586886 with plasma aldosterone level in MESA and demonstrated that global deletion of TASK-1 channels in mice produces a mild-hyperaldosteronism, not associated with a decrease in renin. Our results suggest that genetic variation in the KCNK3 gene may contribute to BP variation and less severe hypertensive disorders in which aldosterone may be one of several causative factors.