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Functional ₁-Adrenoceptor Subtypes in Human Submandibular Glands

¹ Department of Oral and Maxillofacial Surgery and
⁴ Department of Oral Pathology, Peking University School and Hospital of Stomatology, Zhong Guan Cun South St. 22, 100081 Beijing, PRC;
² Department of Pathophysiology, Peking University Health Science Center; and
³ Institute of Vascular Medicine, Peking University Third Hospital, Beijing, PRC

View larger version (169K): [in a new window]   Figure 1. The gene imaging and protein expression of 1-adrenoceptor subtypes in human submandibular glands. RT-PCR products from human submandibular glands were eletrophoresed on 2% agarose gels and visualized with ethidium bromide. M, Marker (100-bp DNA ladder; the lightest band is 500 bp). (A,B,C) 1A-, 1B-, and 1D-adrenoceptor gene expression, respectively. Lane 1 in A, B, and C indicates RT-PCR product amplified from human 1-adrenoceptor subtype full-length cDNA, as a positive control. Lane 2 in A, B, and C indicates RT-PCR product without using reverse-transcriptase, as a negative control. Lane 3 in A, B, and C indicates RT-PCR product amplified from human submandibular gland. (D,E,F) RT-PCR products of 1A-, 1B-adrenoceptor and ß-actin amplified from individuals. (G) Comparison of the relative amounts of transcripts for 1A-adrenoceptor subtype mRNA with 1B-adrenoceptor subtype mRNA. Data were expressed as mean ± SEM (n = 10, P < 0.05). (H,I) 1A- and 1B-adrenoceptor gene expression by in situ hybridization (paraffin section), respectively. Intracytoplasmic hybridization signal was observed in acinar and ductal cells. (J,K) 1A- and 1B-adrenoceptor protein expression by immunohistochemistry (frozen section), respectively. Intracytoplasmic and membrane staining was seen in acinar and ductal cells. (Arrow a indicates acinar cells, arrow d indicates ductal cells.) (L,M) 1A- and 1B-adrenoceptor protein expression by Western blotting. Intensively immunoreactive bands of 55 kDa and 60 kDa, corresponding to the 1A- and 1B-adrenoceptors, were detected. ’Cont’ indicates the positive control, with HEK293 cells transfected with the cDNAs encoding 1-adrenoceptor subtypes. Lanes 1, 2, and 3 in L and M, respectively, indicate the expression of 1A- and 1B-adrenoceptors in human submandibular glands from three persons.

View larger version (18K): [in a new window]   Figure 2. Effects of 1-adrenoceptor subtype selective agonists and antagonist on [Ca2+]i in human submandibular gland cells. (A,C,E) Fluorescence image of fluo-3AM-loaded cells before treatment with phenylephrine, A61603, or 5-methylurapidil. No change in [Ca2+]i fluorescence intensity (FI) was observed after pre-treatment with 5-methylurapidil (E). There were no marked differences in FI among A, C, and E. The maximum of FI was observed at about 1500 sec after agonist treatment. (B,D) A significant increase in [Ca2+]i FI at the individual cell level after treatment with phenylephrine (1 µM) or A61603 (100 µM), respectively. (F) A mild increase in [Ca2+]i FI at the individual cell level with 1 µM of phenylephrine after pre-treatment with 5-methylurapidil (0.1 µM). (G) The dynamics of FI at the individual cell level treated with phenylephrine, A61603, or 5-methylurapidil plus phenylephrine in the human submandibular gland cells. (H) Fluorescence image of fluo-3AM-loaded cells with KCl stimulation (50 mM).