Th. Hendriks

Biochemical aspects of the visual process. XXV. Light-induced calcium movements in isolated frog rod outer segments

Abstract Suspensions of isolated rod outer segments are shown to have a high calcium content of up to 11 moles calcium per mole rhodopsin. Osmotic lysis of the outer segments demonstrates the presence of two calcium fractions, a soluble one and a particulate one. The particulate fraction apparently coincides with the rod disks or with disk fragments. Illumination of intact rod outer segments in calcium-free ATP-containing Ringer solution has no measurable effect upon their total caclium content, but causes a significant shift of calcium from the particulate to the soluble fraction. This light effect is retained in lysed outer segments resuspended in calcium-free ATP-containing Ringer. These results support a function of calcium as a transmitter in light transduction in rod outer segments.

Divergent patterns of matrix metalloproteinase activity during wound healing in ileum and colon of rats.

BACKGROUND: Uncontrolled and increased extracellular matrix degradation during early anastomotic repair in the intestine may reduce wound strength increasing the risk of anastomotic dehiscence. AIMS: To characterise the metalloproteinases present in intact and anastomosed ileum and colon to study their role in matrix degradation after surgery. SUBJECTS: Tissue extracts of uninjured, and of anastomosed rat ileum and colon at postoperative days 1, 2, 3, 7, and 90, were used. METHODS: Metalloproteinases were identified by gelatin and casein zymography. Aminophenylmercuric acetate (APMA) treatment was used to activate latent metalloproteinases. RESULTS: Both uninjured ileum and colon contained a 60 and 67 kDa activity, but a 54 and 72 kDa gelatinase was present in ileum only, and a 51 kDa activity in colon only. APMA treatment converted the 60 kDa protease to 54 and 51 kDa forms and the 72 kDa protease to the 67 kDa form. These gelatinases may correspond to latent and active forms of MMP 1 and MMP 2, respectively. Additional metalloproteinases were observed after anastomotic construction. Both ileum and colon contained 95 and 230 kDa gelatinases, which were converted to 83 and 76 kDa forms by APMA. They may be the latent and active forms of MMP 9, respectively. Gelatinolytic activities of 25 and 28 kDa were only found in anastomosed ileum. Caseinolytic activities were only found in ileum extracts and those were most prominent at day 1, 2, and 3 after surgery. CONCLUSIONS: The metalloproteinase pattern in ileum and colon differ considerably suggesting that matrix degradation after anastomotic construction may also vary.

Does nifedipine suppress atherogenesis in WHHL rabbits

The effects of the calcium antagonist, nifedipine, on atherogenesis were investigated in WHHL rabbits, a unique animal model for human familial hypercholesterolemia. Nifedipine, in a daily dose of 40 mg, was fed orally to 9 rabbits over a period of 26 weeks, resulting in serum concentrations of between 740 and 1370 ng/ml. Rabbits were killed at an age of 40 weeks and atherosclerotic plaque formation in various aortic segments was quantified. Atherosclerosis was most pronounced in the aortic arch and the thoracic aorta, plaques covering, respectively, 59 +/- 17% and 17 +/- 9% of total vessel area. These results are similar to those observed in a control group, which received the same diet and no nifedipine and displayed lesions on 62 +/- 19% and 21 +/- 13% of total area of aortic arch and thoracic aorta, respectively. Although variations in plaque area between WHHL rabbits are large and thus preclude the observation of small effects, the efficacy of nifedipine as an anti-atherogenic agent in rabbits with hereditary hypercholesterolemia appears questionable.

Biochemical aspects of the visual process XXXII. Movement of sodium ions through bilayers composed of retinal and rod outer segment lipids

Abstract The leakage of Na + from sonicated liposomes, composed of rod outer segment lipids, retinal lipids and a 4 : 1 phosphatidylcholine/phosphatidylserine mixture, has been studied. Both retinal and rod outer segment lipid liposomes lose Na + faster than Ca 2+ which indicates that the observed leakage occurs from closed liposomal structures. Liposomes from rod outer segment lipids are extremely leaky, losing sodium about 10 times as fast as retinal lipid liposomes and twice as fast as the phosphatidylcholine/phosphatidylserine liposomes. This high permeability of rod outer segment lipid liposomes, as compared to retinal lipid liposomes, is probably due to both the higher degree of unsaturation of the fatty acid chains and their lower cholesterol content. In the rod outer segment lipid extract 48% of the fatty acid chains consists of docosahexaenoic acid (C 22:6 ) against only 24% in retinal lipid extract. Rod outer segment lipids contain 4.0% cholesterol against 12.3% in retinal lipids. The sodium leakage from rod outer segment lipid liposomes is little affected by the presence of 5 mM calcium in the external dialysis medium, but with the two other types of liposomes significant decreases in permeability of about 20% are observed. The results are discussed in connection with the role of cations in visual excitation.

Movement of calcium through artificial lipid membranes and the effects of ionophores

The calcium efflux from multi-layered vesicles (liposomes) of different lipid composition has been studied. Liposomes composed of lipids extracted from cattle retinas are compared with liposomes which consist of phosphatidylcholine or a 1:1 phosphatidylcholine/phosphatidylserine mixture. The percentages of 45-Ca capture by these three types of liposomes are 10, 1 and 4% respectively; The efflux rates are 2.5-10- minus 6, 2-10- minus 6 and 4-10- minus 5 S- minus 1 respectively. The semilogarithmic efflux curves for phosphatidylcholine and phosphatidylcholine/phosphatidylserine liposomes are linear with time, but those for the retinal lipid liposomes are discontinuous. The activation energy for the calcium efflux from the latter liposomes is about 10.5 kcal/mol, both before and after the discontinuity. The ionophores X537A and A23187 enhance the calcium leakage from retinal lipid liposomes, the latter ionophore being much more effective than the former. At high concentrations both ionophores seem to transport calcium as a 1: 2 Ca-ionophore complex. At low ionophore concentrations, however, X537A appears to transport calcium as a 1:1 complex, but A23187 as a 2:1 complex.

Loss of collagen from experimental intestinal anastomoses: Early events

Collagen lysis, which always occurs to some extent in the wound area, is thought to be the underlying cause for breakdown of intestinal anastomoses. Therefore, we have studied the loss of collagen around ileal and colonic anastomoses in New Zealand White rabbits during the first 48 hr after operation. In the ileum, significant lysis of collagen in the anastomotic area, as represented by a decreased level of hydroxyproline, occurs from 12 hr postoperatively onward. Maximal loss of hydroxyproline, as compared to preoperative values, is 27% measured 24 hr after operation. In the colon, significant lysis of collagen occurs after 3 hr. The lowest level of hydroxyproline measured during the experimental period is found 48 hr after operation, where the concentration is decreased by 38%. Changes in ileum are restricted to the anastomotic area, while in the colon the decrease in hydroxyproline extends along the intestinal wall, particularly in a proximal direction. The fact that total protein concentrations do not vary significantly indicates that the lowered hydroxyproline levels are specific. Microscopic examination of the wound area shows that the cellular response during the first 24 hr after wounding is restricted to granulocytes. It is suggested that granulocyte collagenase is mainly responsible for the observed lysis of collagen after intestinal anastomosis.

Morphometric analysis of the effects of antineoplastic drugs on mucosa of normal ileum and ileal anastomoses in rats

Antineoplastic agents affect the healing of intestinal anastomoses. They often induce anorexia and diarrhea, possibly caused by morphological changes in the small intestinal mucosa. These changes were evaluated in the rat ileum. Animals in group I underwent only intestinal surgery while those in groups II and III underwent surgery on the third day of a 5-day course with cisplatin (in two different doses), bleomycin, and 5-fluorouracil. The parameters were: number of mitoses in crypts, crypt depth, villus height, width, and contour length, measured in the mucosa of primarily resected segments of the ileum and of the anastomotic area. Surgery yields an increased crypt depth and villus length in the anastomotic area without changing villus width. The changes in intestinal crypts precede those in villi. Antineoplastic drugs decrease crypt mitotic rate, villus height, width, and contour length. After cessation of antineoplastic chemotherapy mitotic activity increases. The shallower and shorter villi increase in width and length resulting in an increased villus contour length and area. A linear relation exists between villus contour length and villus height and width. Thus, antineoplastic polychemotherapy, dose-dependently, reduces and surgical trauma increases intestinal proliferative activity. However, the morphologic changes do not unequivocally explain possible metabolic disturbances causing retarded intestinal wound healing.

Biochemical aspects of the visual process XXIV. Adenylate cyclase and rod photoreceptor membranes: A critical appraisal☆

Abstract Adenylate cyclase was found to be present in rod outer segment preparations, but its specific activity was only about 1% of activities reported in earlier studies. In frog activities ranged from 0.015 to 1.1 nmoles 3′,5′ cyclic AMP/mg protein per 10 min depending on the method of preparation and homogenization. In cattle, the rod outer segment layer obtained after sucrose density gradient centrifugation, had an activity of 0.22 nmole 3′,5′ cyclic AMP/mg protein per 10 min. Furthermore a second (more dense) layer obtained in this procedure possessed a 10 times higher specific activity. Light decreased the adenylate cyclase activity in the rod outer segment suspensions of both frog and cattle, but the maximal inhibition was about 50% at extensive illumination. Light did not affect the activity in the second layer, unless rod outer segment layer material was present, indicating that an inhibitory diffusible factor is released from outer segments during illumination. Evidence that either Ca 2+ or free all- trans retinaldehyde constitutes this factor could not be obtained. The activities of some marker enzymes in the two layers and in whole retina homogenates from cattle were determined. Comparison of some properties of the adenylate cyclase activities in the two layers and consideration of these enzyme activities do not exclude the possibilty that the activity in the rod outer segment material is due to contamination with other retinal material. The available evidence does not support a direct role for 3′,5′ cyclic AMP in the visual excitation process.

High-Dose Preoperative Irradiation without Detrimental Effect on Early Repair of Anastomoses in the Colon of the Rat

Preoperative radiotherapy as an adjunct to surgery for rectal carcinoma is generally thought to impair the healing of colorectal anastomoses. To delineate the presumed hazards of preoperative irradiation, we investigated this effect in a new model where, in contrast to experiments reported so far, anastomoses were constructed using normal tissue for the proximal limb and irradiated tissue for the distal limb. A group of 120 male Wistar rats, divided randomly into 12 groups of 10 each, were used. In 60 animals, a colonic segment of 2.2 cm was irradiated with a single dose of 25 Gy X rays administered 28 or 5 days or 3 or 1 day(s) before colonic resection. For each experimental group, a control group was included which was sham-irradiated on the same preoperative day. The animals were sacrificed on the third or the seventh postoperative day, and healing of the anastomosis was evaluated by measurement of bursting pressure, breaking strength and hydroxyproline concentration and content. Comparison between each experimental group and its control group showed that preoperative irradiation did not reduce the strength of the anastomoses. Also, the concentration and content of hydroxyproline in the tissue of the anastomoses were unchanged. These data indicate that construction of a colonic anastomosis consisting of one irradiated bowel end in rats is not by definition detrimental to the development of early wound strength.

Intraperitoneal cytostatics impair early post-operative collagen synthesis in experimental intestinal anastomosesP6.

Collagen synthesis in intestinal anastomoses has been measured in rats after in vivo administration of cytostatics. The cytostatics were administered during 5 consecutive days either intravenously or intraperitoneally. On day 3 of the course the rats received both an ileal and a colonic anastomosis. The animals were sacrificed 3 and 7 days after operation. The cytostatics regimen used was a combination of 5-fluorouracil, bleomycin and cisplatinum in a dose of 10, 2 and 0.35 mg kg-1 day-1, respectively. In an additional group, a twice higher dose was given intraperitoneally. Three days after operation a severe inhibition of the collagen synthesis was observed in all the cytostatics treated groups, both in ileum and in colon. The effects of intraperitoneal administration were much more pronounced than those observed after an equal dose given intravenously. Seven days after operation the collagen synthesis in the intravenously treated groups was restored to the level of the control group. However, in the intraperitoneal groups the collagen synthesis in ileal anastomoses was still inhibited. Thus, cytostatics suppress collagen synthesis in intestinal anastomoses. The effect is more severe after intraperitoneal than after intravenous administration, confirming our earlier hypothesis that the former mode of administration comprises a higher risk for anastomotic integrity.