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. 2010 Jun;58(6):499-507.
doi: 10.1369/jhc.2009.955047. Epub 2009 Nov 24.

Localization of ATP-sensitive K+ channel subunits in rat submandibular gland

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Localization of ATP-sensitive K+ channel subunits in rat submandibular gland

Ming Zhou et al. J Histochem Cytochem. 2010 Jun.

Abstract

ATP-sensitive K(+) (K(ATP)) channel subunits were investigated in rat submandibular gland (SMG). RT-PCR detected the presence of mRNA transcripts of the Kir6.1, Kir6.2, SUR2A, and SUR2B in the SMG, whereas SUR1 mRNA was barely detected. Western blot analysis provided the evidence that these four K(ATP) channel subunits are expressed in rat SMG. Immunostaining detected that these four K(ATP) channel subunits are widely distributed, with different intensities, in myoepithelial cells, epithelial cells of intercalated ducts, granular convoluted tubules, striated ducts, and excretory ducts. Immunofluorescence double staining showed that Kir6.1 and Kir6.2 colocalized with SUR2A in the myoepithelial cells, granular convoluted tubules, striated ducts, and excretory ducts. Kir6.1 and Kir6.2 also colocalized with SUR2B, mainly in the duct system, e.g., the granular convoluted tubules, striated ducts, and excretory ducts. Taken together, these results indicate that the K(ATP) channels in SMG may consist of Kir6.1, Kir6.2, SUR2A, and SUR2B, with various combinations of colocalization with each other, and may play important roles in rat SMG during salivary secretion.

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Figures

Figure 1
Figure 1
RT-PCR of rat submandibular gland (SMG) and skeletal muscle (SKM) total RNA. The 1.5% agarose gels show bands of DNA fragments amplified with primer pairs specific for Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B, as well as glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Primers were tested to amplify fragments of 212 bp for Kir6.1, 297 bp for Kir6.2, 558 bp for SUR1, 155 bp for SUR2A (primers for SUR2A only), 328 bp for SUR2A, 152 bp for SUR2B (primers for both SUR2A and SUR2B), and 393 bp for G3PDH. The result shows that all ATP-sensitive K+ channel subunits, but not SUR1, are expressed in the SMG, compared with the positive control of the SKM RNA.
Figure 2
Figure 2
Immunoblot analysis of Kir6.1, Kir6.2, SUR2A, and SUR2B from rat SMG. Anti-Kir6.1 antibody recognized a prominent polypeptide of ∼50 kDa with a dimer of ∼100 kDa (Lane Kir6.1), and anti-Kir6.2 antibody recognized a ∼48-kDa polypeptides (Lane Kir6.2) in the SMG. Anti-SUR2A antibody recognized a remarkable polypeptide of ∼140 kDa (Lane SUR2A), and anti-SUR2B antibody revealed a polypeptide of ∼120 kDa (Lane SUR2B) in the SMG.
Figure 3
Figure 3
(A) Immunoreactivity with anti-Kir6.1 was observed to be weak in intercalated ducts (Ic) and moderate in the granular convoluted tubules (G) and striated ducts (St), and faint immunoreactivity was also seen in the myoepithelial cells (arrow). (B) Immunoreactivity with anti-Kir6.1 was observed to be moderate in the excretory ducts (Ex). Smooth muscle of blood vessel also showed weak immunoreactivity for Kir6.1 (Bv). (C) Immunoreactivity with anti-Kir6.2 was observed to be weak in the intercalated ducts (Ic) and moderate in granular convoluted tubules (G) and striated ducts (St). Myoepithelial cells showed faint immunoreactivity with anti-Kir6.2 (arrow). (D) Immunoreactivity with anti-Kir6.2 was observed to be moderate in excretory ducts (Ex). Blood vessels (Bv) also showed weak immunoreactivity of Kir6.2. Bar = 20 μm.
Figure 4
Figure 4
(A) Immunoreactivity with anti-SUR2A was observed to be faint in myoepithelial cells (arrow), weak in intercalated ducts (Ic), moderate in granular convoluted tubules (G), and moderate to intense in striated ducts (St). (B) Immunoreactivity of SUR2A was observed to be intense in the excretory duct (Ex) and weak in the smooth muscles of blood vessels (Bv). (C) Immunoreactivity with anti-SUR2B was observed to be faint in myoepithelial cells (arrow), weak in the intercalated ducts (Ic), and moderate in granular convoluted tubules (G) and striated ducts (St). (D) Immunoreactivity of SUR2B was observed as moderate to intense in the striated (St) and excretory ducts (Ex) and weak in the blood vessels (Bv). Bar = 20 μm.
Figure 5
Figure 5
Immunofluorescence double staining shows the expression of Kir6.1 and Kir6.2 in the SMG as green (Alexa 488, A,D,G,J), SUR2A and SUR2B as red (Alexa 594, B,E,H,K). The merged images (C,F,I,L) indicate that SUR2A colocalizes with Kir6.1 (C) and/or with Kir6.2 (F), and that SUR2B colocalizes with Kir6.1 (I), and/or with Kir6.2 (L) in the myoepithelial cells (thin arrows), granular convoluted tubules (G), striated ducts (St), and excretory ducts (Ex), as well as blood vessels (bold arrows). Bar = 50 μm.

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