4. Adenosine (0-18 mM), adenosine monophosphate (0-28 mM), or adenosine triphosphate (0 19 mM) lowered CA secretion evoked by transmural stimulation by about 40 %, but had no effect on secretion induced by ACh.5. Isoprenaline (4-52 #UM), propranolol (11 58 ,M), clonidine (13-00 ,SM), phenoxybenzamine (3-30 /M), and 4-aminopyridine (3 mM) did not modify CA secretion evoked by transmural stimulation or by ACh.6. Perfusion of the adrenal gland with 0-25 mM-Ca-Krebs solution completely abolished CA secretion evoked by transmural stimulation, but ACh-induced secretion was still 30-50 % of the control value. 20 mM-Mg blocked electrically induced secretion by 60 %, but that evoked by ACh was unaffected.7. Perfusion with Ca-free Krebs solution for 2 hr did not completely abolish the response. However, treatment with EGTA (5 mM) for 30 min totally blocked ACh-induced secretion.8. La or Mn were more effective in blocking transmurally evoked secretion than ACh-evoked secretion ofCA. Verapamil (0-1 mM) had no significant effect on secretion evoked by either procedure. A 5-fold increase in its concentration caused about 75 % blockade of secretion.9. Differential effects of various ions and agents on CA secretion are explained on the basis that these compounds affect neurosecretory properties of the presynaptic splanchnic nerve terminals and of chromaffin cells differently.
Triadin is an intrinsic membrane protein first identified in the skeletal muscle junctional sarcoplasmic reticulum and is considered to play an important role in excitation-contraction coupling. Using polyclonal antibodies to skeletal muscle triadin, we have identified and characterized three isoforms in rabbit cardiac muscle. The cDNAs encoding these three isoforms of triadin have been isolated by reverse transcription-polymerase chain reaction and cDNA library screening. The deduced amino acid sequences show that these proteins are identical in their N-terminal sequences, whereas the C-terminal sequences are distinct from each other and from that of skeletal muscle triadin. Based upon both the amino acid sequences and biochemical analysis, all three triadin isoforms share similar membrane topology with skeletal muscle triadin. Immunofluorescence staining of rabbit cardiac muscle with antibodies purified from the homologous region of triadin shows that cardiac triadin is primarily confined to the I-band region of cardiac myocytes, where the junctional and corbular sarcoplasmic reticulum is located. Furthermore, we demonstrate that the conserved region of the luminal domain of triadin is able to bind both the ryanodine receptor and calsequestrin in cardiac muscle. These results suggest that triadin colocalizes with and binds to the ryanodine receptor and calsequestrin and carries out a function in the lumen of the junctional sarcoplasmic reticulum that is important for both skeletal and cardiac muscle excitation-contraction coupling.
Tryptic digestion of tyrosine hydroxylase (TH) isolated from rat adrenal glands labeled with 32Pi produced five phosphopeptides. Based on the correspondence of these phosphopeptides with those identified in TH from rat pheochromocytoma cells, four phosphorylation sites (Ser8, Ser19, Ser31, and Ser40) were inferred. Field stimulation of the splanchnic nerves at either 1 or 10 Hz (300 pulses) increased 32P incorporation into TH. At 10 Hz, the phosphorylation of Ser19 and Ser40 was increased, whereas at 1 Hz, Ser19, Ser31, and Ser40 phosphorylation was increased. Stimulation at either 1 or 10 Hz also increased the catalytic activity of TH, as measured in vitro (pH 7.2) at either 30 or 300 microM tetrahydrobiopterin. Nicotine (3 microM, 3 min) increased Ser19 phosphorylation, vasoactive intestinal polypeptide (10 microM, 3 min) increased Ser40 phosphorylation, and muscarine (100 microM, 3 min) increased TH phosphorylation primarily at Ser19 and Ser31. Vasoactive intestinal polypeptide, but not nicotine or muscarine, mimicked the effects of field stimulation on TH activity. Thus, the regulation of rat adrenal medullary TH phosphorylation by nerve impulses is mediated by multiple first and second messenger systems, as previously shown for catecholamine secretion. However, different sets of second messengers are involved in the two processes. The action of vasoactive intestinal polypeptide as a secretagogue involves the mobilization of intracellular calcium, whereas its effects on TH phosphorylation are mediated by cyclic AMP. This latter effect of vasoactive intestinal polypeptide and the consequent increase in Ser40 phosphorylation appear to be responsible for the rapid activation of TH by splanchnic nerve stimulation.
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