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.

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The purpose of the present study was to determine the molecular mechanism of stimulatory actions of ACh and vasoactive intestinal polypeptide (VIP) by determining the role of various second messengers in the neurohumoral secretion. Toward such a goal, we measured cAMP, cGMP, protein kinase (PKC) activity, 3H-inositol triphosphate (3H-IP3), and 45Ca uptake in the adrenal medulla subjected to various treatments. Stimulation of splanchnic nerve endings increased 45Ca uptake, cAMP content, 3H-IP3, and PKC activity in the adrenal medulla. If muscarinic receptors of chromaffin cells were selectively activated by perfusion with muscarine, 3H-IP3 content and PKC activity were enhanced. Nicotine, on the other hand, increased only 45Ca uptake without affecting any other second messenger. Perfusion with VIP increased PKC activity and cAMP and 3H-IP3 content. None of the procedures affected cGMP content. Interplay among various second messengers was further investigated by studying interactions of nicotinic, muscarinic, and VIP-ergic receptors in modulation of catecholamine (CA) secretion and by using agents known to activate specific second messengers (e.g., forskolin, phorbol esters). Our results show that muscarine, VIP, and phorbol ester facilitated nicotine-evoked secretion by increasing PKC activity, and it was associated with an additional increase in 45Ca accumulation. On the other hand, secretion evoked by nicotine as well as muscarine was facilitated by forskolin without additional increase in 45Ca accumulation. A novel feature of the study is that ACh and VIP activate three types of receptors on chromaffin cells to stimulate and mutually facilitate the secretion of CA by generating various second messengers.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of phorbol ester, phorbol 12,13-dibutyrate, was investigated on the overflow of tritium from 3H-noradrenaline-loaded sympathetic neurons of the isolated perfused salivary gland of the rat. Stimulation (1 Hz for 60 s)-evoked overflow of tritium was enhanced by phorbol ester. A significant enhancement was seen at 1 nmol/l, which increased to a maximum level (over 4-fold) at 30 nmol/l. The spontaneous overflow of radioactivity, however, was not affected by any concentration of phorbol ester. The facilitatory effect of phorbol ester on stimulation-evoked overflow was observed in the presence of inhibitors of neuronal and extraneuronal uptake as well as after removal of negative feedback inhibition of release by presynaptic alpha-adrenoceptors. Tyramine (7 mumol/l for 10 min) caused a marked increase in the overflow of tritium in either the presence or absence of calcium. However, tyramine-induced overflow was not enhanced by phorbol ester. It is concluded that protein kinase C of sympathetic neurons is involved in an exocytotic release of the transmitter.