Natriuretic peptides and nitric oxide stimulate cGMP synthesis in different cellular compartments.

Cyclic nucleotide-gated (CNG) channels are a family of ion channels activated by the binding of cyclic nucleotides. Endogenous channels have been used to measure cyclic nucleotide signals in photoreceptor outer segments and olfactory cilia for decades. Here we have investigated the subcellular localization of cGMP signals by monitoring CNG channel activity in response to agonists that activate either particulate or soluble guanylyl cyclase. CNG channels were heterologously expressed in either human embryonic kidney (HEK)-293 cells that stably overexpress a particulate guanylyl cyclase (HEK-NPRA cells), or cultured vascular smooth muscle cells (VSMCs). Atrial natriuretic peptide (ANP) was used to activate the particulate guanylyl cyclase and the nitric oxide donor S-nitroso-n-acetylpenicillamine (SNAP) was used to activate the soluble guanylyl cyclase. CNG channel activity was monitored by measuring Ca2+ or Mn2+ influx through the channels using the fluorescent dye, fura-2. We found that in HEK-NPRA cells, ANP-induced increases in cGMP levels activated CNG channels in a dose-dependent manner (0.05-10 nM), whereas SNAP (0.01-100 microM) induced increases in cGMP levels triggered little or no activation of CNG channels (P < 0.01). After pretreatment with 100 microM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, ANP-induced Mn2+ influx through CNG channels was significantly enhanced, while SNAP-induced Mn2+ influx remained small. In contrast, we found that in the presence of IBMX, both 1 nM ANP and 100 microM SNAP triggered similar increases in total cGMP levels. We next sought to determine if cGMP signals are compartmentalized in VSMCs, which endogenously express particulate and soluble guanylyl cyclase. We found that 10 nM ANP induced activation of CNG channels more readily than 100 muM SNAP; whereas 100 microM SNAP triggered higher levels of total cellular cGMP accumulation. These results suggest that cGMP signals are spatially segregated within cells, and that the functional compartmentalization of cGMP signals may underlie the unique actions of ANP and nitric oxide.

Glutamate receptors expressed in {\it Xenopus\/} oocytes: Studies in function and regulation

The amino acid glutamate is the primary excitatory neurotransmitter for the CNS and is responsible for the majority of fast synaptic transmission. Glutamate receptors have been shown to be involved in multiple forms of synaptic plasticity such as LTP, LTD, and the formation of specific synaptic connections during development. In addition to contributing to the plasticity of the CNS, glutamate receptors also are involved in, at least in part, various pathological conditions such as epilepsy, ischemic damage due to stroke, and Huntington's chorea. The regulation of glutamate receptors, particularly the ionotropic NMDA and AMPA/KA receptors is therefore of great interest. In this body of work, glutamate receptor function and regulation by kinase activity was examined using the Xenopus oocyte which is a convenient and faithful expression system for exogenous proteins. Glutamate receptor responses were measured using the two-electrode voltage clamp technique in oocytes injected with rat total forebrain RNA. NMDA elicited currents that were glycine-dependent, subject to block by Mg$\sp{2+}$ in a voltage-dependent manner and sensitive to the specific NMDA antagonist APV in a manner consistent with those types of responses found in neural tissue. Similarly, KA-evoked currents were sensitive to the specific AMPA/KA antagonist CNQX and exhibited current voltage relationships consistent with the calcium permeable type II KA receptors found in the hippocampus. There is evidence to indicate that NMDA and AMPA/KA receptors are regulated by protein kinase A (PKA). We explored this by examining the effects of activators of PKA (forskolin, 1-isobutyl-3-methylxanthine (IBMX) and 8-Br-cAMP) on NMDA and KA currents in the oocyte. In buffer where Ca$\sp{2+}$ was replaced by 2 mM Ba$\sp{2+},$ forskolin plus IBMX and 8-Br-cAMP augmented currents due to NMDA application but not KA. This augmentation was abolished by pretreating the oocytes in the kinase inhibitor K252A. The use of chloride channel blockers resulted in attenuation of this effect indicating that Ba$\sp{2+}$ influx through the NMDA channel was activating the endogenous calcium-activated chloride current and that the cAMP mediated augmentation was at the level of the chloride channel and not the NMDA channel. This was confirmed by (1) the finding that 8-Br-cAMP increased chloride currents elicited via calcium channel activation while having no effect on the calcium channels themselves and (2) the fact that lowering the Ba$\sp{2+}$ concentration to 200 $\mu$M abolished the augmentation NMDA currents by 8-Br-cAMP. Thus PKA does not appear to modulate ionotropic glutamate receptors in our preparation. Another kinase also implicated in the regulation of NMDA receptors, calcium/phospholipid-dependent protein kinase (PKC), was examined for its effects on the NMDA receptor under low Ba$\sp{2+}$ (200 $\mu$M) conditions. Phorbol esters, activators of PKC, induced a robust potentiation of NMDA currents that was blockable by the kinase inhibitor K252A. Furthermore activation of metabotropic receptors by the selective agonist trans-ACPD, also potentiated NMDA albeit more modestly. These results indicate that neither NMDA nor KA-activated glutamate receptors are modulated by PKA in Xenopus oocytes whereas NMDA receptors appear to be augmented by PKC. Furthermore, the endogenous chloride current of the oocyte was found to be responsive to Ba$\sp{2+}$ and in addition is enhanced by PKA. Both of these latter findings are novel. In conclusion, the Xenopus oocyte is a useful expression system for the analysis of ligand-gated channel activity and the regulation of those channels by phosphorylation. ^

Regulation of sodium/glucose cotransporter expression in LLC-PK$\sb1$ cells

Expression of the Na$\sp+$/glucose cotransporter (SGLT1), a differentiated function of the pig kidney epithelial cell line LLC-PK$\sb1$ derived from proximal tubule, was further investigated. The differentiation inducer hexamethylene bisacetamide (HMBA) and IBMX, an inhibitor of cAMP phosphodiesterase, each stimulated a significant increase in Na$\sp+$/glucose cotransport activity, levels of the 75 kD cotransporter subunit and steady-state levels of the SGLT1 message. The action of HMBA is associated with involvement of polyamines and protein kinase C, and is synergistic with cAMP. We provide evidence that cAMP-elevating agents increase Na$\sp+$/glucose cotransporter expression, at least in part, via a post-transcriptional mechanism. Two molecular species of SGLT1 mRNA (3.9 kb and 2.2 kb) are transcribed from the same gene in LLC-PK$\sb1$ cells and differ only in the length of the 3$\sp\prime$ untranslated region (3$\sp\prime$ UTR). cAMP elevation differentially stabilized the 3.9 kb SGLT1 transcript from degradation but not the 22 kb species. UV-cross-linking and label transfer experiments indicated that cyclic AMP elevation was associated with formation of a 48 kD protein complex with a specific domain within the 3$\sp\prime$ UTR of SGLT1 mRNA. The binding was competitively inhibited by poly (U) and other U-rich RNA species such as c-fos ARE, and modulated by a protein kinase A-mediated phosphorylation/dephosphorylation mechanism. The binding site was mapped to a 120-nucleotide 3$\sp\prime$ UTR sequence which contains a uridine-rich region (URE). Our study provides the first demonstration that renal SGLT1 is post-transcriptionally regulated by a phosphorylation/dephosphorylation mechanism, and provides a deeper insight into gene regulation of this physiologically important cotransporter. ^