No abstract

A human T-cell line producing human T-cell leukemia virus type I (HTLV-I), MT-2, was injected intravenously into female F344 rats aged 5 weeks to make HTLV-I carrier rats. Antibody against HTLV-I was detected at the 5th week after MT-2 injection, and its titer reached a high plateau which continued from the 15th to the 27th week. The antibodies were against p19, p24, p28 and p53 of HTLV-I antigens from MT-2 cells. The gag, pX and LTR nucleotide sequences of HTLV-I provirus were demonstrated by using polymerase chain reaction (PCR) in the peripheral-blood mononuclear cells of 3 rats at the 44th week and 2 at the 66th to 68th week out of 8 F344 rats injected with MT-2 cells. Quantification of the HTLV-I proviral sequence revealed that 30 to 60 molecules were present in 10(5) peripheral-blood mononuclear cells, indicating that the rats were chronically infected with HTLV-I. HTLV-I-infected rats could serve as a small-animal model for studying the pathophysiological state of HTLV-I carriers and also that of HTLV-I infection on various HTLV-I-related diseases, including adult T-cell leukemia and HTLV-I-associated myelopathy.

String-shaped reconstituted smooth muscle (SM) fibers were prepared in rectangular wells by thermal gelation of a mixed solution of collagen and cultured SM cells derived from guinea pig stomach. The cells in the fiber exhibited an elongated spindle shape and were aligned along the long axis. The fiber contracted in response to KCl (140 mM), norepinephrine (NE; 10(-7) M), epinephrine (10(-7) M), phenylephrine (10(-6) M), serotonin (10(-6) M), and histamine (10(-5) M), but not acetylcholine (10(-5) M). Phentolamine (10(-7) M) produced a parallel rightward shift of the NE dose-response curve. Moreover, NE-induced contraction was partially inhibited by nifedipine and completely abolished by the intracellular Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester, the myosin light chain kinase inhibitor ML-9, the Rho kinase inhibitor Y-27632, and papaverine. A [(3)H]quinuclidinyl benzilate binding study revealed that the loss of response to acetylcholine was due to the loss of muscarinic receptor expression during culture. The expression of contractile proteins in the fibers was similar to that in cultured SM cells. These results suggest that, although the fiber is not a model for fully differentiated SM, contractile mechanisms are maintained.

1 In Ca-free solution, the contractile response of guinea-pig taenia caecum to 1O-'M carbachol was mediated through muscarinic receptors and was reduced time-dependently by desensitization with 1O-4M carbachol, but not 1O-4M histamine. On the other hand, the response to 1O-4M histamine was shown to be mediated through HI-receptors and to be reduced time-dependently by desensitization with either iO-4 M histamine or 10' M carbachol. 2 The maximal K+ contraction was not changed by desensitization with carbachol or histamine. Thus, contractile proteins and voltage-dependent Ca channels maintain their normal functions. 3 To study the coupling of Ca channel activity in cell surface membrane to receptor activation, the contractile responses elicited by carbachol and histamine added simultaneously with Ca to Ca-free solution were measured. The response elicited by carbachol plus Ca was not changed by desensitization with carbachol, while that elicited by histamine plus Ca was reduced by desensitization with histamine. These results show that desensitization by carbachol occurs at a post-receptor site, whereas that induced by histamine occurs at H,-receptors. 4 After desensitization with carbachol, but not histamine, the contractile response to 5 x 1O 2M caffeine in Ca-free solution was significantly reduced. 5 These results show that short-term desensitization of guinea-pig taenia caecum by carbachol is heterologous and occurs at intracellular Ca stores, while that induced by histamine is homologous and occurs at histamine Hl-receptors.

To characterize the functional differentiation of neural stem cells into smooth muscle cells, multipotent stem cells in the central nervous system (CNS) were isolated from rat embryonic day 14 (E14) cortex and cultured by neurosphere formation in serum‐free medium in the presence of 10 ng ml−1 of basic fibroblast growth factor. Differentiation was induced by the addition of 10 % fetal bovine serum to low‐density cultures (2.5 × 103 cells cm−2). Immunological analyses and reverse transcriptase‐polymerase chain reaction indicated that the differentiated cells expressed smooth‐muscle‐specific marker proteins such as SM‐1, SM‐2, and SMemb myosin heavy chains, SM‐22, basic calponin and α‐smooth‐muscle actin, but not the astrocyte marker glial fibrillary acidic protein. To examine whether smooth‐muscle‐like cells that are differentiated from CNS stem cells possess the characteristics of contractile smooth muscle, we prepared reconstituted collagen gel fibres and measured their contractile tension. The reconstituted fibres were prepared by thermal gelation of collagen and the differentiated cells. The fibres contracted in response to treatment with KCl (80 mm), ACh (100 μm), endothelin‐1 (10 nm), endothelin‐2 (10 nm), and prostaglandin F2α (100 μm). ACh‐induced contraction was partially inhibited by the L‐type voltage‐dependent Ca2+ channel inhibitor nifedipine and by the intracellular Ca2+ chelator 1,2‐bis (2‐aminophenoxy) ethane‐N,N,N’,N'‐tetraacetic acid acetoxymethyl ester, the myosin light chain kinase inhibitor ML‐9, the Rho kinase inhibitor Y‐27632, dibutyryl cAMP and 8‐bromo‐cGMP. These results suggest that CNS stem cells give rise to smooth muscle cells in vitro that have an identical contractile function to smooth muscle in vivo.