Impaired relaxation of stomach smooth muscle in mice lacking cyclic GMP-dependent protein kinase I

Abstract

1. Guanosine 3', 5'-cyclic monophosphate (cyclic GMP)-dependent kinase I (cGKI) is a major receptor for cyclic GMP in a variety of cells. Mice lacking cGKI exhibit multiple phenotypes, including severe defects in smooth muscle function. We have investigated the NO/cGMP- and vasoactive intestinal polypeptide (VIP)/adenosine 3', 5'-cyclic monophosphate (cyclic AMP)-signalling pathways in the gastric fundus of wild type and cGKI-deficient mice. 2. Using immunohistochemistry, similar staining patterns for NO-synthase, cyclic GMP- and VIP-immunoreactivities were found in wild type and cGKI-deficient mice. 3. In isolated, endothelin-1 (3 nM - 3 microM)-contracted, muscle strips from wild type mice, electrical field stimulation (1 - 16 Hz) caused a biphasic relaxation, one initial rapid, followed by a more slowly developing phase. In preparations from cGKI-deficient mice only the slowly developing relaxation was observed. 4. The responses to the NO donor, SIN-1 (10 nM - 100 microM), and to 8-Br-cyclic GMP (10 nM - 100 microM) were markedly impaired in strips from cGKI-deficient mice, whereas the responses to VIP (0.1 nM - 1 microM) and forskolin (0.1 nM - 1 microM) were similar to those in wild type mice. 5. These results suggest that cGKI plays a central role in the NO/cGMP signalling cascade producing relaxation of mouse gastric fundus smooth muscle. Relaxant agents acting via the cyclic AMP-pathway can exert their effects independently of cGKI.

Figure 1

The gastrointestinal tract at autopsy of 5 week-old wild type (left) and litter-matched cGKI-deficient mouse (right). Arrows indicate the pylorus and the caecum of the cGKI-deficient mouse.

Figure 2

Immunofluorescence images of the gastrointestinal wall from the gastric fundus of wild type (a,c,e) and cGKI-deficient (b,d,f) mice. (a) and (b) demonstrate cGKI-immunoreactivity, (c) and (d) demonstrate NOS-immunoreactivity, and (e) and (f) demonstrate VIP-immunoreactivity. Bar 100 μm.

Figure 3

Immunofluorescence images demonstrating cyclic GMP-immunoreactivity in the gastrointestinal wall from the gastric fundus of wild type (a,b) and cGKI-deficient (c,d) mice. (a) and (c) demonstrate the immunoreactivity after preincubation of sodium nitroprusside, whereas (b) and (d) without. Bar 100 μm.

Figure 4

Original tracings showing electrical field stimulation (supramaximal voltage, 0.5 ms pulses, varying frequency 1–16 Hz)-induced relaxations of the gastric fundus. (a) wild type mice. (b) cGKI-deficient mice.

Figure 5

(Upper part) The first relaxant phase of the response to electrical field stimulation (supramaximal voltage, 0.5 ms pulses, varying frequency 1–16 Hz) nerves in smooth muscle strips from the mouse gastric fundus. Wild type mice: n=6; cGKI-deficient mice: n=6. (Lower part) The second relaxant phase of the response to electrical field stimulation (supramaximal voltage, 0.5 ms pulses, varying frequency 1–16 Hz) nerves in smooth muscle strips from the mouse gastric fundus. Wild type mice: n=6; cGKI-deficient mice: n=6. ^*P<0.05.

Figure 6

(Upper part) SIN-1-induced relaxation of smooth muscle strips from in the mouse gastric fundus. Wild type mice: n=6; cGKI-deficient mice: n=6: ^*P<0.05. (Lower part) 8-Br-cyclic GMP-induced relaxation of smooth muscle strips from the gastric fundus. Wild type mice: n=6; cGKI-deficient mice: n=6. ^***P<0.001.

Figure 7

(Upper part) Forskolin-induced relaxation of smooth muscle strips from the gastric fundus. Wild type mice: n=6; cGKI-deficient mice: n=6. (Lower part) VIP-induced relaxation of smooth muscle strips from the gastric fundus. Wild type mice: n=6; cGKI-deficient mice: n=6. ^*P<0.05.