Mixed lineage kinases DLK (dual leucine zipper-bearing kinase) and MLK3 have been proposed to function as mitogen-activated protein kinase kinase kinases in pathways leading to stress-activated protein kinase/c-Jun NH 2 -terminal kinase activation. Differences in primary protein structure place these MLK (mixed lineage kinase) enzymes in separate subfamilies and suggest that they perform distinct functional roles. Both DLK and MLK3 associated with, phosphorylated, and activated MKK7 in vitro. Unlike MLK3, however, DLK did not phosphorylate or activate recombinant MKK4 in vitro. In confirmatory experiments performed in vivo, DLK both associated with and activated MKK7. The relative localization of endogenous DLK, MLK3, MKK4, and MKK7 was determined in cells of the nervous system. Distinct from MLK3, which was identified in non-neuronal cells, DLK and MKK7 were detected predominantly in neurons in sections of adult rat cortex by immunocytochemistry. Subcellular fractionation experiments of cerebral cortex identified DLK and MKK7 in similar nuclear and extranuclear subcellular compartments. Concordant with biochemical experiments, however, MKK4 occupied compartments distinct from that of DLK and MKK7. That DLK and MKK7 occupied subcellular compartments distinct from MKK4 was confirmed by immunocytochemistry in primary neuronal culture. The dissimilar cellular specificity of DLK and MLK3 and the specific substrate utilization and subcellular compartmentation of DLK suggest that specific mixed lineage kinases participate in unique signal transduction events.A large body of work has focused on signal transduction via protein kinases generically termed mitogen-activated protein kinases (MAPK) 1 that link a variety of extracellular signals to cellular responses as diverse as proliferation, differentiation, and apoptosis (reviewed in Refs. 1-3). Biochemical and genetic evidence has demonstrated that activation of a prototypical MAPK occurs through sequential activation of a series of upstream kinases: a serine/threonine MAPK kinase kinase (MAP-KKK) phosphorylates a dual specificity protein kinase (MAPKK or MKK or MEK) that in turn phosphorylates and activates a MAPK. Three groups of mammalian MAPKs and the upstream kinases and stimuli that activate them have been studied most extensively. These include the p42/p44 MAPK s (extracellular signal-regulated kinases, ERK1 and ERK2), that are generally activated by mitogens and differentiation inducing stimuli, the p46/p54 SAPK s (stress-activated protein kinases, SAPKs), and the p38 MAPK s. Stress-activated protein kinases were discovered as the principal c-Jun NH 2 -terminal kinases and therefore have also been termed JNKs. Distinct from ERK1 and ERK2, the SAPKs are predominantly activated by cell stress-inducing signals such as heat shock, ultraviolet irradiation, proinflammatory cytokines, hyperosmolarity, ischemia/reperfusion, and axonal injury.Like previously identified MAPK pathways in mammalian cells and yeast, the SAPK pathways were initially thought to lead in a lin...

The purpose of this study was to systematically investigate the abundance of each of the adenosine receptor subtypes in the preglomerular microcirculation vs. other vascular segments and vs. the renal cortex and medulla. Rat preglomerular microvessels (PGMVs) were isolated by iron oxide loading followed by magnetic separation. For comparison, mesenteric microvessels, segments of the aorta (thoracic, middle abdominal, and lower abdominal), renal cortex, and renal medulla were obtained by dissection. Adenosine receptor protein and mRNA expression were examined by Western blotting, Northern blotting, and RT-PCR. Our results indicate that compared with other vascular segments and renal tissues, A1 and A2B receptor protein and mRNA are abundantly expressed in the preglomerular microcirculation, whereas A2A and A3 receptor protein and mRNA are barely detectable or undetectable in PGMVs. We conclude that, relative to other vascular and renal tissues, A1 and A2B receptors are well expressed in PGMVs, whereas A2A and A3 receptors are notably deficient. Thus A1 and A2B receptors, but not A2A or A3 receptors, may importantly regulate the preglomerular microcirculation.

Abstract-Hypertrophied cardiac myocytes exhibit prolonged action potentials and decreased transient outward potassium current (I to

Cardiac myocytes respond to ␣ 1 -adrenergic receptor stimulation by a progressive hypertrophy accompanied by the activation of many fetal genes, including skeletal muscle ␣-actin. The skeletal muscle ␣-actin gene is activated by signaling through an MCAT element, the binding site of the transcription enhancer factor-1 (TEF-1) family of transcription factors. Previously, we showed that overexpression of the TEF-1-related factor (RTEF-1) increased the ␣ 1 -adrenergic response of the skeletal muscle ␣-actin promoter, whereas TEF-1 overexpression did not. Here, we identified the functional domains and specific sequences in RTEF-1 that mediate the ␣ 1 -adrenergic response. Chimeric TEF-1 and RTEF-1 expression constructs localized the region responsible for the ␣ 1 -adrenergic response to the carboxyl-terminal domain of RTEF-1. Site-directed mutagenesis was used to inactivate eight serine residues of RTEF-1, not present in TEF-1, that are putative targets of ␣ 1 -adrenergicdependent kinases. Mutation of a single serine residue, Ser-322, reduced the ␣ 1 -adrenergic activation of RTEF-1 by 70% without affecting protein stability, suggesting that phosphorylation at this serine residue accounts for most of the ␣ 1 -adrenergic response. Thus, these results demonstrate that RTEF-1 is a direct target of ␣ 1 -adrenergic signaling in hypertrophied cardiac myocytes.Cardiac myocytes respond to ␣ 1 -adrenergic receptor stimulation by a progressive hypertrophy (1), accompanied by a characteristic reactivation of many fetal genes, including ␤-myosin heavy chain (2), skeletal muscle ␣-actin (SKA) 1 (3, 4), and brain natriuretic factor (5). The ␣ 1 -adrenergic stimulation of these promoters requires an MCAT element (4, 5), with the sequence CATN(T/C)(T/C) (6). Transcription factors of the transcription enhancer factor-1 (TEF-1) multigene family bind to MCAT elements in the promoters of many genes expressed in cardiac and skeletal muscle cells (7). Thus, a role for TEF-1-related transcription factors in mediating the ␣ 1 -adrenergic response has been proposed (4,8,9). Previously, we showed that the TEF-1-related factor RTEF-1 could potentiate the ␣ 1 -adrenergic stimulation of the ␤-myosin heavy chain and SKA promoters, when overexpressed in cardiac myocytes. In contrast, TEF-1 did not affect their response to ␣ 1 -adrenergic stimulation. Thus, we proposed a role for RTEF-1 in mediating the ␣ 1 -adrenergic reactivation of fetal genes in cardiac myocytes (10).The different effects of TEF-1 and RTEF-1 overexpression on the ␣ 1 -adrenergic response of the SKA promoter must reflect differences in how TEF-1 and RTEF-1, or their associated co-factors, are modified by ␣ 1 -adrenergic signaling. TEF-1 and RTEF-1 are identical in their DNA binding domains, are highly conserved in their carboxyl-terminal activation domains, but diverge in sequences flanking the DNA binding domain (see Fig. 1). To what extent these divergent sequences confer functional differences was not known.In the present study, we took advantage of the different response of...

Hypertension in spontaneously hypertensive rats (SHRs) is due in part to enhanced effects of vasoactive peptides on the renal vasculature. We hypothesize that the G i signal transduction pathway enhances renovascular responses to vasoactive peptides in SHRs more so than in normotensive Wistar-Kyoto (WKY) rats. To test this hypothesis, we examined in isolated perfused kidneys from SHRs and WKY rats the renovascular responses (assessed as changes in renal perfusion pressure in mm Hg) to angiotensin II (10 nM) and vasopressin (3 nM) in the presence and absence of UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; an agonist that selectively activates the G i pathway by stimulating ␣ 2 -adrenoceptors]. In SHR, but not WKY, kidneys, UK-14,304 (10 nM) enhanced (P Ͻ 0.05) renovascular responses to angiotensin II (control WKY, 43 Ϯ 6; UK-14,304-treated WKY, 52 Ϯ 19; control SHR, 66 Ϯ 17; UK-14,304-treated SHR, 125 Ϯ 16) and vasopressin (control WKY, 42 Ϯ 17; 36 Ϯ 11; control SHR, 16 Ϯ 8; 83 Ϯ 17). Pretreatment of SHRs with pertussis toxin (30 g/kg, intravenously, 3-4 days before study) to inactivate G i blocked the effects of UK-14,304. Western blot analysis of receptor expression in whole kidney and preglomerular microvessels revealed similar levels of expression of AT 1 , V 1a , and ␣ 2A receptors in SHRs compared with WKY rats. We conclude that activation of ␣ 2 -adrenoceptors selectively enhances renovascular responses to angiotensin II and vasopressin in SHRs via an enhanced cross talk between the G i signal transduction pathway and signal transduction pathways activated by angiotensin II and vasopressin.The spontaneously hypertensive rat (SHR) is a widely employed model of human genetic hypertension; however, the pathophysiology of hypertension in SHRs has eluded four decades of intensive investigation. Nonetheless, progress has been made, and it is now clear that hypertension in SHRs requires the vasoactive peptide angiotensin II (Ang II) (Bunkenburg et al., 1991;Lee et al., 1991); yet SHRs do not have increased circulating (Shiono and Sokabe, 1976) or tissue concentrations of Ang II (Campbell et al., 1995). It is also apparent that the SHR kidney is critical to the hypertensive state in this genetic model. In this regard, transplantation studies indicate that hypertension tracks the SHR kidney (Rettig and Unger, 1991). A third requirement for full expression of hypertension in SHRs is an intact G i signal transduction pathway. Blockade of the G i signal transduction pathway with a single dose of pertussis toxin causes a prolonged antihypertensive response in adult SHRs and delays the development of hypertension in young SHRs (Li and Anand-Srivastava, 2002). To understand the pathophysiology of hypertension in the SHR, the challenge is to propose and test hypotheses that reconcile these seemingly unrelated findings.We hypothesize that hypertension in SHRs is caused in part by an augmented cross talk in the renal microcirculation between the G i signal transduction pathway and the si...

The "extracellular cAMP-adenosine pathway" refers to the conversion of cAMP to AMP by ecto-phosphodiesterase, followed by metabolism of AMP to adenosine by ecto-5Ј-nucleotidase, with all the steps occurring in the extracellular compartment. This study investigated whether the extracellular cAMP-adenosine pathway exists in proximal tubules. Freshly isolated proximal tubules rapidly converted basolaterally administered cAMP to AMP and adenosine. Proximal tubular cells in culture (first passage) rapidly converted apically administered cAMP to AMP and adenosine. In both freshly isolated proximal tubules and cultured proximal tubular cells, conversion of cAMP to AMP and adenosine was affected by a broadspectrum phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine), an ecto-phosphodiesterase inhibitor (1,3-dipropyl-8-p-sulfophenylxanthine), and a blocker of ecto-5Ј-nucleotidase (␣,␤-methyleneadenosine-5Ј-diphosphate) in a manner consistent with exogenous cAMP being processed by the extracellular cAMP-adenosine pathway. In cultured proximal tubular cells, but not freshly isolated proximal tubules, stimulation of adenylyl cyclase increased extracellular concentrations of cAMP, AMP, and adenosine plus inosine, and these changes were also modulated by the inhibitors in a manner consistent with the extracellular cAMP-adenosine pathway. Conversion of renal interstitial (basolateral) cAMP and AMP to adenosine in vivo was shown by microdialysis coupled with ion trap mass spectrometry. Western blot analysis showed A 1 , A 2A , and A 3 receptors on both apical and basolateral proximal tubular membranes, with A 1 and A 2A receptors more highly expressed on basolateral compared with apical membranes. We conclude that cAMP that reaches either the apical or basolateral membranes of proximal tubular cells is converted in part to adenosine that has ready access to adenosine receptors.

Adenosine regulates tubular transport in collecting ducts (CDs); however, the sources of adenosine that modulate ion transport in CDs are unknown. The extracellular cAMP-adenosine pathway refers to the conversion of cAMP to AMP by ectophosphodiesterase, followed by metabolism of AMP to adenosine by ecto-5Ј-nucleotidase, with all steps occurring in the extracellular compartment. The goal of this study was to assess whether the extracellular cAMP-adenosine pathway exists in CDs. Studies were conducted in both freshly isolated CDs and in CD cells in culture (first passage) that were derived from isolated CDs. Purity of CDs was confirmed by microscopy, by Western blotting for aquaporin-1, aquaporin-2, bumetanidesensitive cotransporter type 1, and thiazide-sensitive cotransporter; and by reverse transcription-polymerase chain reaction for adenosine receptors. Both freshly isolated CDs and CD cells in culture converted exogenous cAMP to AMP and adenosine. In both freshly isolated CDs and CD cells in culture, conversion of cAMP to AMP and adenosine was affected by a broadspectrum phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine), an ectophosphodiesterase inhibitor (1,3-dipropyl-8-psulfophenylxanthine), and a blocker of ecto-5Ј-nucleotidase (␣,␤-methylene-adenosine-5Ј-diphosphate) in a manner consistent with exogenous cAMP being processed by the extracellular cAMP-adenosine pathway. In CD cells in culture, stimulation of adenylyl cyclase increased extracellular concentrations of cAMP, AMP, and adenosine, and these changes were also modulated by the aforementioned inhibitors in a manner consistent with the extracellular cAMP-adenosine pathway. In conclusion, the extracellular cAMP-adenosine pathway is an important source of adenosine in CDs.Several lines of evidence indicate that adenosine importantly regulates ion transport in collecting duct (CD) epithelial cells: 1) A 1 receptors are more highly expressed in CDs compared with other nephron segments (Smith et al., 2001); 2) in A6 cells, a model system for transport in the CD and in collecting ducts adenosine receptor agonists alter ion transport (Lang et al

Abstract-TheKey Words: receptors Ⅲ neuropeptides Ⅲ peptides Ⅲ hypertension T he renin-angiotensin system (RAS) is essential for the development and maintenance of genetic hypertension in spontaneously hypertensive rats (SHRs). 1,2 Moreover, transplantation studies reveal that, in addition to the RAS, the SHR kidney is pivotal to the pathophysiology of hypertension in the SHR. 3,4 Finally, the renal sympathetic nervous system also appears to importantly contribute to the pathophysiology of hypertension in SHRs. In support of this latter concept, chronic denervation of the SHR kidney both delays the development of hypertension and attenuates the maximum increase in blood pressure in SHR. [5][6][7][8] Thus, there appears to be a coinvolvement of the RAS, the sympathetic nervous system, and the kidney in SHR hypertension.Many studies have been performed in search of a possible explanation for the coinvolvement of the RAS and the kidney in SHR hypertension. In this regard, studies do not support an increased expression of renal angiotensin II (Ang II) receptors 9 or increased levels of circulating 10 or renal 11 Ang II; also, SHRs do not have altered renal Ang II degradation rates. 12 However, SHRs do exhibit increased renovascular responses to Ang II, 13,14 and this appears to be the explanation for the coinvolvement of the RAS and the kidney in SHR hypertension.Our previous research indicates that G i mediates, in part, the enhanced renovascular response to Ang II in SHR. For example, pertussis toxin, an inhibitor of G i , abolishes the increased renovascular response to Ang II in SHR. 15,16 Importantly, activation of renal sympathetic nerves leads to the release of neuropeptide Y (NPY). 17 NPY is an example of a pancreatic polypeptide (PP)-fold peptide, 18 and NPY binds with high affinity to Y 1 , Y 2 ,