1. We investigated whether adrenomedullary chromaffin cells (AMCs) derived from neonatal (postnatal day (P) 1-P2) and juvenile (P13-P20) rats, and maintained in short-term culture (1-3 days), express 02-chemoreceptive properties. 2. In whole-cell recordings, the majority (-70%; n = 47) of neonatal

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In mammals, ventilatory acclimatization to hypoxia is associated with an enhanced chemosensitivity of the O2-sensing carotid body, resulting in an increased respiratory drive. To test whether this sensitization involves long-term modulation of ion channel function in endogenous O2 chemoreceptors, i.e., type 1 cells, we exposed cultures of dissociated rat carotid body to chronic hypoxia (6% O2) for 1–2 weeks, before monitoring the electrophysiological properties of type 1 cells using whole-cell, perforated patch recording. Chronic hypoxia augmented voltage-dependent inward Na+ and Ca2+ currents in type 1 cells, without significant changes in voltage dependence of activation or steady-state inactivation. However, after normalizing for the concomitant increase in cell size, indicated by the whole-cell capacitance, only the Na+ current density was significantly enhanced. The Na+ current was sensitive to tetrodotoxin (TTX; 0.5–1 microM) or choline substitution, whereas most of the Ca2+ current was sensitive to the L-type calcium channel blocker, nifedipine (10 microM). Several of these effects of hypoxia were mimicked qualitatively by growing normoxic cultures in the presence of agents that elevate intracellular cyclic AMP, including dibutyryl cAMP (db-cAMP; 200 microM-1 mM) and forskolin (10 microM); treatment with similar concentrations of dibutyryl cyclic GMP was ineffective. Na+ channel induction by db-cAMP was abolished by the protein synthesis inhibitor, cycloheximide (90–180 microM). In current- clamp mode, these altered chemoreceptors had typical resting potentials of approximately -55 mV, and following depolarization often fired multiple spikes that appeared to consist of both short-duration Na+ and long-duration Ca2+ components. We propose that chronic hypoxia, acting in part through cAMP-dependent pathways, increases electrical excitability and calcium mobilization in type 1 cells, and these adaptations may help enhance chemosensitivity during hypoxic acclimatization.

Chronic hypoxia sensitizes the ventilatory reflex in mammals and causes enlargement of the carotid body, a peripheral arterial chemosensory organ. To investigate possible underlying mechanisms, in the absence of circulatory changes, we exposed cultures of dissociated rat carotid body containing the oxygen sensors (i.e., chromaffin-like glomus cells) to chronic hypoxia (6% 02) over a period of 2 weeks. After a delay of a few days, the Na+ current density in hypoxia-treated glomus cells increased gn ntly, reaching values up to 6 times that seen in normoxic (20% 02) controls.In addition the whole-cell capacitance, an indicator of cell size, was also significantly larger (3-4 times control) in glomus cells exposed to chronic hypoxia. Both effects were mimicked qualitatively by chronic treatment of normoxic cultures with N6,02-dibutyryladenosine 3',S'-cycic monophosphate, but not nerve growth factor, which is known to induce similar changes in the chromaffm cell line PC12. Thus, the physiological and morphological effects of chronic hypoxia on the carotid body in vivo may be due in part to a cAMP-mediated stimulation of Na+ channel expression and hypertrophy in the chemosensory glomus cells.Conditions of chronic hypoxia, as occur in humans and animals living at high altitude (1) or in patients with hypoxic lung disease (2), cause enlargement or hypertrophy of the carotid body, an organ that senses blood Po2 and controls ventilation. In addition, there is a time-dependent sensitization of this chemosensory pathway (3, 4), resulting in an increased respiratory drive that is thought to be important during acclimatization to hypoxia. The above structural and functional changes may be mediated via a direct action of low arterial Po2 on the carotid-body oxygen sensors (i.e., the chromaffin-like glomus cells), which contain a unique class of 02-sensitive K+ channels (5-7), or indirectly, by way of blood-borne factors arising elsewhere in the circulation. Though there is evidence that the hypertrophic response of the carotid body during chronic hypoxia is accompanied in part by an increased size of the glomus cells (8, 9), the underlying mechanisms associated with the morphological and physiological adaptations of the carotid body to chronic hypoxia are largely unknown. Moreover, these mechanisms are especially difficult to investigate in vivo, where circulatory factors cannot be readily controlled or eliminated.This laboratory has been investigating (7, 10, 11) chemotransduction mechanisms by using dissociated cell cultures of the rat carotid body in which the chemosensory glomus cells survive for several weeks and are accessible for electrophysiological patch-clamp/whole-cell recording. The ability to control the cellular, fluid, and gaseous environments in these cultures allowed us to investigate whether direct exposure ofcarotid body cells alone to chronic hypoxia could alter the physiological and morphological properties of the glomus cells, in the absence of extraneous circulatory factors. In this study, we com...

In the present study, we investigate the role of specific cytoplasmic tail (CT) regions of the D1A receptor in mediating dopamine (DA)-induced phosphorylation, desensitization and endocytosis. Results obtained in human embryonic kidney (HEK) cells expressing the wild-type (WT) or truncation forms (D425, D379 and D351) of the D1A receptor show that sequences located downstream of Gly379 regulate DA-mediated phosphorylation-dependent desensitization of D1A receptors. However, the longer truncation mutant D351 failed to undergo detectable DA-induced phosphorylation while exhibiting DA-induced desensitization features similar to the shorter truncation mutant D379. These data potentially suggest a novel role for a receptor phosphorylation-independent process in the DA-promoted D1A subtype desensitization. Our immunofluorescence data also suggest that sequences located between Cys351 and Gly379 play an important role in DA-mediated receptor endocytosis. Additionally, time-course studies were done in intact cells expressing WT or truncation receptors to measure the observed rate constant for adenylyl cyclase (AC) activation or k obs , a parameter linked to the receptor-G protein coupling status. In agreement with the desensitization data, D425-and D379-expressing cells exhibit an increase of k obs in comparison with WT-expressing cells. Nevertheless, D351-expressing cells, which harbor similar desensitization features of D379-expressing cells, display no change in k obs when compared with WT-expressing cells. Our results suggest that a defective DA-induced endocytosis may hamper D351 resensitization and concomitant increase in k obs . Thus, our study showing that specific D1A receptor CT sequences regulate DA-induced phosphorylation, desensitization, and endocytosis highlights the underlying molecular complexity of signaling at dopaminergic synapses.

Using dissociated carotid body (OB) cultures prepared from neonatal (postnatal days 5-7; P7) orjuvenile (postnatal day 19-20; P20) rats, we compared catecholaminergic properties and mechanisms of 02 sensing in glomus cells grown in normoxic (Nox; 20% 02) and chronically hypoxic (CHox; 6% 02) environments for up to 2 weeks. In Nox cultures, basal dopamine(DA) release, determined by HPLC and normalized to the number of tyrosine hydroxylase-positive glomus cells present, was similar for P7 and P20 cultures (~-~0.3 pmol/1 ‚000 cells! 15 mm) and was unaffected by culture duration (2 vs. 12 days). Acute hypoxia (5 and 10% 02) caused a dosedependent stimulation (6>< and 3>< basal, respectively) in DA release, that was inhibited by nifedipine (10 ‚aM). DA release was also stimulated by high extracellular K( 30 mM) and iberiotoxin (200 nM), a selective blocker of Po 2-regulated, Ca-dependent K~channel in glomus cells. The stimulatory effect of iberiotoxin was similar to 5% 02 in P20 cultures, but substantially less (about onehalf) in P7 cultures. In contrast, in CHox cultures, basal DA release was substantially elevated, -~8xNox levels, although this did not correlate with significant differences in stores. Further, whereas acute hypoxia (5% 02) and high K~also stimulated DA release in CHox cultures (~2xand~3x basal), iberiotoxin (200 nM) did not. Thus, after chronic hypoxia in vitro, there is an enhanced basal catecholamine release and an apparent down-regulation of functional Ca-dependent K~channels in OB chemoreceptors. These cellular adaptations may relate to changes in OB chemosensitivity during chronic hypoxemia. Key Words: Glomus cell culture-Chronic hypoxia in vitro-Dopamine-Catecholamine release-HPLCIberiotoxin-sensitive K + channels.

To delineate the role of the cytoplasmic tail in the distinct binding and coupling properties of human dopamine D1-like receptors, chimeric receptors were generated in which the entire tail region of wild-type human D1A (or D1) and D1B (or D5) receptors was exchanged. The hD1A-D1BT, but not hD1B-D1AT, receptor expression was dramatically reduced compared with wild-type receptor expression. Swapping the cytoplasmic tail resulted in a full switch of dopamine binding affinity and constitutive activity, while dopamine potency decreased and agonist-mediated maximal activation of adenylyl cyclase increased for both chimeras. Hence, the cytoplasmic tail plays a crucial role in D1-like receptor expression, agonist binding affinity and constitutive activation but regulates in a distinct fashion the formation of D1A and D1B receptor active states upon dopamine binding.z 2000 Federation of European Biochemical Societies.

The 'cross-talk' between different types of neurotransmitters through second messenger pathways represents a major regulatory mechanism in neuronal function. We investigated the effects of activation of protein kinase C (PKC) on cAMPdependent signaling by structurally related human D1-like dopaminergic receptors. Human embryonic kidney 293 (HEK293) cells expressing D1 or D5 receptors were pretreated with phorbol-12-myristate-13-acetate (PMA), a potent activator of PKC, followed by analysis of dopamine-mediated receptor activation using whole cell cAMP assays. Unpredictably, PKC activation had completely opposite effects on D1 and D5 receptor signaling. PMA dramatically augmented agonist-evoked D1 receptor signaling, whereas constitutive and dopamine-mediated D5 receptor activation were rapidly blunted. RT-PCR and immunoblotting analyses showed that phorbol ester-regulated PKC isozymes (conventional: a, bI, bII, c; novel: d, e, g, h) and protein kinase D (PKCl) are expressed in HEK293 cells. PMA appears to mediate these contrasting effects through the activation of Ca 2+ -independent novel PKC isoforms as revealed by specific inhibitors, bisindolylmaleimide I, Gö 6976, and Gö 6983. The finding that cross-talk between PKC and cAMP pathways can produce such opposite outcomes following the activation of structurally similar D1-like receptor subtypes is novel and further strengthens the view that D1 and D5 receptors serve distinct functions in the mammalian nervous and endocrine systems.