Male guinea pigs, 400 to 500 g body weight, have been divided into three groups: a control one, which received no treatment, a second one, comprising of animals, receiving treatment with CoCl₂, and a third one, including animals which have been injected with the same volume of saline. The animals from the second group received a subcutaneous injection of CoCl₂, diluted in 1 ml saline in concentration 80 mg kg^-1 b.w., 22 to 24 hours before the euthanasia. On the day of the experiment animals have been killed by a stun on the neck, decapitation and exsanguination. Smooth muscle strips were cut from either half of the stomach wall from the fundic region and the circular layer of the tissue was excised for isometric recording of the contractile force as well as for isolation of single cells for the patch clamp studies. The other half of the stomach fundus was kept in whole and further fixed for immunochistochemical studies (see below). Animals used for these experiments were picked up from the first and the second groups. The group, used to account for the effect of the injection itself on HO-1 induction, comprised of animals used for biochemical studies only.

For immunohistochemical investigation 16 guinea-pigs (8 control and 8 treated with CoCl₂) were killed as described above. The stomach’s fundus were cut in several slabs and were immediately fixed by immersion with 4% paraformaldehyde and 0.15% picric acid in 0.1 mol L^-1 phosphate buffer (PB), pH 7.3 for 24 h at 4^oC. Some of the pieces were processed for paraffin embedding. Tissue sections (5 µm thick) were mounted on chrom alum-coated slides, deparaffinized in xilol, rehydrated in a graded series of ethanol and washed in 0.05 mol.L^-1 phosphate buffered saline (PBS). Other slabs were washed over night in 20% sucrose solution in 0.1 mol L^-1 PBS. Consecutive 30 µm thick sections from muscle coat of the fundus were prepared on “Reichert Yung” freezing microtome and collected in solution of sucrose. They were washed overnight in PBS after several changes.

The color pictures of the sections obtained were taken via digital camera, and the intensity of the staining into the smooth muscle layer was analyzed using Corel Draw software. The data obtained from HO-1 stained sections were compared between treated and non-treated animals as follows. The intensity of the staining of at least 20 different sections prepared from each animal was taken as an average after subtracting the background staining separately for each section. Background staining was defined as an immunonegative staining that could be observed in the areas of the vascular lumen. After performing such a normalizing procedure for the sections taken from each animal, the differences in the staining intensity between treated and non-treated animals were statistically evaluated. The intensity of HO-2 staining was assessed by comparing the average intensity of the non-specific staining obtained as a background taken from selected immunonegative areas of each section with the staining of the smooth muscle layer. Comparison for the staining intensity between different sections was not performed. For the purpose of such analysis only 30 µm thick sections were used.

Contraction experiments. The preparations for contractile studies were done according to the previously described procedure. After excision fundic strips (10 mm long) were mounted vertically in 10 ml organ baths and stretched under tension of 10 mN. The organ baths were filled with Ca-free Krebs’ solution (see below) and strips were left for 30 min to relax. The bath solution was then changed to 2.5 mmol.L^-1 Ca-containing Krebs to initiate contractions, and the strips were left to equilibrate for 45–60 min until a stable spontaneous contractile activity developed. The bath solutions were kept in dark, thermostatically controlled at 37 °C and continuously bubbled with a gas mixture (95% O₂ and 5% CO₂). During the equilibrium period the bath solution was changed every 20 min. The spontaneous isometric contractions of preparations were registered using Grass Instruments force-displacement transducers (capacitance ± 10 g) and recorded on paper line recorder (type MTA 175, Kutesz, Hungary). The neurally independent tone of the tissue was evaluated in the presence of 1 µmol L^-1 tetrodotoxin to block the release of neurotransmitters. The responses of the strips to field electrical stimulation (FES) were registered in the presence of atropine (1 µmol L^-1) and L-^ωNitro-L-arginine and evoked on the plateau of the PgF_2α induced tone. Parameters of stimulation were as follows: 2 Hz and 5 Hz, at 50 V, for 30 sec, applied via platinum electrodes placed into the bath chamber at 2.5 mm distance from both sides of the strips. The relaxatory responses of the strips to electrical stimulation were estimated as a percentage of the maximal tone developed before addition of CaCl₂ as compared to the basal level of the tone as measured in Ca²⁺-free Krebs following the stretching. The data were assessed for statistical significance using paired Tukey-Kramer Multiple Comparison Test at p < 0.05.

Krebs’ solution, used in contraction experiments, consisted of (in mmol.L^-1): 110 NaCl, 6 KCl, 1.2 MgCl₂, 1.2 NaH₂PO₃, 11 glucose, 25 NaHCO₃, 2.5 CaCl₂, which was bubbled with 95% O₂ and 5% CO₂ gas mixture to achieve a pH of 7.4 at 37 °C.

All substances used for solutions contraction experiments were obtained from ICN Biomedicals, Irvine, CA, USA, except NaOH (for pH adjustment), vitamin E and atropine sulphate, which were SIGMA products. Hemin and Sn-protoporphyrin IX were purchased from ALEXIS Biochemicals, Switzerland.

In the untreated animals, immunohistochemical studies revealed the presence of NADPH-d-positive neurons (Figure 1). The staining is in single neurons from the ganglia of the myentheric plexus, with weak staining in the smooth muscle tissue. In the treated guinea-pigs, the majority of neurons in the myenteric ganglia of the gastric fundus are NADPH-positive, with the staining mainly on the bodies of the neurons and the neural pathways not stained. In addition, smooth muscle tissues are also intensely stained, indicating a large amount of NADH-d in these tissues.

Immunochistochemically stained 30 µm thick sections of the guinea pig stomach fundus for non-treated and treated animals. Circles (A and B) shows myenteric ganglia. White arrows (C and D) point to immunopositive nerve cell bodies in the myenteric ganglia. Black arrows (E and F) point to immunonegative cell bodies.

Immunohistochemical studies for HO-1 revealed the presence of immunopositive neurons mainly in the treated animals (Figure 2), which is indicative of the induction of the enzyme in the tissues examined.

Figure 1. 

Immunohistochemistry of NADPH-d.

Figure 2. 

Immunohistochemistry of HO-1 and HO-2. Immunohistochemically stained 30 µm thick sections of the guinea pig stomach fundus for non-treated and treated animals.

Pretreating the strips of untreated animals with PgF_2α in the presence of atropine (1 µmol L-1) and L-NNA (0.1 mmol L-1) resulted in increased muscle tone. After its stabilization, a 2Hz and 5Hz FES, each of 30 sec duration, was applied. After the first stimulation, the re-stabilization of muscle tone (Figure 3a) was awaited. Blocking NOS resulted in a 76% increase in the effect of Hemin on the FES-evoked response at a stimulation frequency of 2 Hz (Figure 4). The presence of VitE in the solution reduces the effect of Hemin by up to 49%. At 5Hz, the increase in Hemin‘s effect is 51%, and the presence of VitE reduces it to 41%. On the other hand, blocking NOS results in a 23% (±7) effect of SnPPIX (Table 1). The presence of Hemin results in a 34% reduction in the effect of SnPPIX. At high stimulation frequency (5Hz), blocking has no significant effect on the effect of SnPPIX.

Treatment of experimental animals with CoCl₂ did not affect the spontaneous contractile activity and tone of the preparations. The increase in the tone of the preparations in the presence of atropine (1 µmol L-1) and L-NNA (0.1 mmol L-1) before electrical stimulation takes into account the longer time required to stabilize the tone after PgF_2α, instability, as well as changing the configuration of the relaxer response (Figure 3b).

After relative stabilization of muscle tone, a 2Hz and 5Hz FES, each lasting 30 s, was administered. After the end of the first stimulation, a re-stabilization of muscle tone was awaited.

Hemin has an effect on the relaxation response only at low stimulation frequency – 2Hz, and blocking NOS increases its effect by 18% (Figure 5). However, the presence of VitE in the solution increases the Hemin effect by up to 61%. Blocking NOS eliminates the effect of SnPPIX (Table 1). Treatment with Hemin before SnPPIX increases the amplitude of the relaxation response. At 5Hz, blocking the NOS has no significant effect on the effect of SnPPIX.

Figure 3. 

(a) Original trace representing the effect of hemin (0.01 µmol.L^-1) on the FES-evoked relaxation in non-treated guinea-pig gastric fundus strips. (b) Original trace representing the effect of hemin (0.01 µmol.L^-1) on the FES-evoked relaxation in treated guinea-pig gastric fundus strips.  – adding PgF_2α (0.5 ηmol.L^-1) to evoke contractile response. Incubation with hemin is for 20 min., with Vitamin E – 10 min. before hemin application. The responses of the strips to EFS were registered in the presence of atropine (1 µmol.L^-1).

Figure 4. 

FES-evoked relaxations in non-treated and treated guinea pigs. (a) Effect of Hemin (10 µmol.L^-1) on 2 Hz (n = 5) evoked relaxations. (b) Effect of Hemin (10 µmol.L^-1) on 5 Hz (n = 5) induced relaxatory responses of the strips. Data are the means ±SEM of experiments (* p < 0.5).

Figure 5. 

FES-evoked relaxations in non-treated and treated guinea pigs in presence of VitE (100 µmol L^-1). (a) Effect of Hemin (10 µmol L^-1) on 2 Hz (n = 5) evoked relaxations. (b) Effect of Hemin (10 µmol L^-1) on 5 Hz (n = 5) induced relaxatory responses of the strips. Data are the means ±SEM of experiments (* p < 0.5).