Diabetes mellitus: polymorphonuclear leukocyte (PMN) filtration parameters and PMN membrane fluidity after chemotactic activation.

The goal of this research was to determine leukocyte rheology at baseline and after chemotactic activation in type I and type II diabetics. In 19 normal subjects, 21 type I diabetics, and 16 type II diabetics at baseline and after in vitro chemotactic activation (prolonged for 5 and 15 minutes) with two stimulating agents (4-phorbol 12-myristate 13-acetate [PMA] and N-formyl-methionyl-leucyl-phenylalanine [fMLP]), we evaluated polymorphonuclear (PMN) filtration parameters (using a St. George filtrometer [Carri-Med, Dorking, UK] and considering the initial relative flow rate [IRFR] and the concentration of clogging particles [CP]) and PMN membrane fluidity (obtained by marking PMNs with the fluorescent probe 1-(4-[trimethylamino]phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). At baseline, there was a difference between normals and type I and II diabetics for PMN membrane fluidity only. After activation in normals and diabetics of both types, a significant variation was present in PMN filtration parameters (IRFR and CP) at both 5 and 15 minutes. In normals, no variation was present in PMN membrane fluidity after activation with PMA or fMLP. After PMN activation, only in type I diabetics was a significant decrease in PMN membrane fluidity present at both 5 and 15 minutes. After PMN activation with either PMA or fMLP in comparison to basal values, only the mean variation (delta%) of the IRFR was significantly different between normals, type I diabetics, and type II diabetics at both 5 and 15 minutes. From the data obtained, it is evident that after activation, the PMN filtration pattern shows a specific behavior in diabetics of both types, while PMN membrane fluidity changes only in type I diabetics. The latter finding may be the basis of a metabolic pattern present in PMNs of this type, revealed after in vitro activation.

Chronic renal failure: leukocyte rheology and polymorphonuclear cytosolic Ca2+ concentration.

In a group of subjects with chronic renal failure (CRF), we evaluated the leukocyte rheological activity, expressed as the filtration rate, the polymorphonuclear (PMN) membrane fluidity and the PMN cytosolic Ca2+ content. Using the St. George's Filtrometer, we determined the filtration rate of unfractionated, mononuclear and PMN leukocytes. Using the fluorescent probe 1.4-(trimethylamino)-phenyl-4-phenyl-hexatriene (TMA-DPH), we examined the PMN membrane fluidity and, using the Fura 2-AM, the PMN cytosolic Ca2+ content. From the results obtained, it is evident that only the initial relative flow rate of unfractionated leukocytes was significantly reduced in subjects with CRF, while the filtration parameters of mononuclear and PMN cells did not distinguish normals from CRF subjects. No variation was evident for the PMN membrane fluidity, while the PMN cytosolic Ca2+ content was significantly increased in CRF subjects. In these subjects no correlation was found between leukocyte filtration parameters, PMN membrane fluidity, PMN cytosolic Ca2+ content and plasma parameters (blood urea nitrogen and serum creatinine), reflecting the degree of the CRF. In conclusion, in CRF subjects the abnormality of the leukocyte flow properties was restricted to the initial flow rate of unfractionated leukocytes, whereas the increase of PMN cytosolic Ca2+ content might be attributed to PMN activation.

Polymorphonuclear leukocyte membrane fluidity and cytosolic Ca2+ concentration in diabetes mellitus.

We evaluated polymorphonuclear membrane (PMN) fluidity in 32 subjects with type 1 diabetes mellitus, 38 subjects with type 2 diabetes mellitus and 38 normal control subjects, by marking intact and unstimulated PMN cells with the fluorescent probe 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). We also evaluated PMN cytosolic Ca2+ content by marking intact and unstimulated PMN cells with the fluorescent probe Fura 2-AM. PMN membrane fluidity differentiated normal subjects from type 1 and 2 diabetic subjects. The PMN cytosolic Ca2+ concentration did not discriminate type 1 and 2 diabetic subjects from normal control subjects. No statistical correlation was found between PMN membrane fluidity and PMN cytosolic Ca2+ concentration in any of the groups of subjects, nor were significant correlations found between PMN membrane fluidity and cytosolic Ca2+ concentration in several plasma parameters (serum glucose, cholesterol and triglycerides). In conclusion, in type 1 and 2 diabetic patients we found a decrease in PMN membrane fluidity and this decrease, which was greater in type 2 diabetic patients, may be a marker of PMN dysfunction.

Type II diabetics with macrovascular complications: polymorphonuclear leukocyte (PMN) filtration, PMN membrane fluidity and cytosolic Ca2+ content after activation.

We evaluated polymorphonuclear (PMN) filtration parameters, membrane fluidity and cytosolic Ca2+ content in 21 normal subjects and in 18 type II diabetics with macrovascular complications (MVC). Evaluations were carried out at baseline and after in vitro activation prolonged for 5 and 15 min. PMA (4-phorbol 12-myristate 13-acetate) and fMLP (N-formyl-methionyl-leucyl-phenylalanine) were used as stimulating agents. TMA-DPH (1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene) was used as fluorescent probe for the membrane fluidity tests and Fura 2-AM for the cytosolic Ca2+ content. A significant variation was evident in PMN filtration parameters at 5 and 15 min. No variation was present in PMN membrane fluidity and cytosolic Ca2+ content in normals. In type II diabetics with MVC, we found an increase solely in PMN cytosolic Ca2+ content after PMA activation and an early decrease in PMN membrane fluidity and a late increase in PMN cytosolic Ca2+ content after fMLP activation. After PMA activation alone (at 15 min), PMN filtration distinguishes normals from type II diabetics with MVC. The PMN filtration parameters behave similarly in the two groups, but PMN membrane fluidity and cytosolic Ca2+ content behave differently.

Essential hypertension: leukocyte rheology and polymorphonuclear cytosolic Ca2+ content at baseline and after activation.

In 24 hypertensives we evaluated, at baseline, the leukocyte filtration parameters (using the St. George's Filtrometer), polymorphonuclear (PMN) membrane fluidity (with the fluorescent probe 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene [TMA-DPH]) and PMN cytosolic Ca2+ content (with the fluorescent probe Fura 2-AM). In a subgroup of hypertensives (n = 17) the PMN filtration parameters, PMN membrane fluidity and cytosolic Ca2+ content were evaluated after in vitro chemotactic activation (prolonged for 5 and 15 min) with two stimulating agents (4-phorbol 12-myristate 13-acetate [PMA] and N-formyl-methionyl-leucyl-phenylalanine [fMLP]). It was evident, from the baseline data, that there was a significant difference in the mononuclear (MN) initial relative flow rate (IRFR), clogging rate (CR) and clogging particles (CP), and in PMN cytosolic Ca2+ content. There were, however, no differences in the filtration parameters of unfractionated leukocytes and PMNs or in PMN membrane fluidity. After activation, in normals and in hypertensives, a significant variation in PMN filtration parameters was evident. In normals no variation was present in PMN membrane fluidity or cytosolic Ca2+ content after activation. In hypertensives, however, we found an increase solely in PMN cytosolic Ca2+ content after fMLP activation. After PMN activation (at 15 min) one parameter (IRFR) of PMN filtration distinguished normal subjects from hypertensives. No difference between the two groups was found in PMN membrane fluidity or PMN cytosolic Ca2+ content after PMN activation.

Polymorphonuclear leukocyte membrane fluidity and cytosolic Ca2+ content in different clinical conditions.

The aim of the study was to evaluate the polymorphonuclear leukocyte (PMN) membrane fluidity and PMN cytosolic Ca2+ content in several clinical conditions: diabetes mellitus, vascular atherosclerotic disease (VAD), chronic renal failure (CRF), essential hypertension (EH). In 13 subjects with insulin-dependent diabetes mellitus (IDDM), in 24 subjects with non-insulin-dependent diabetes mellitus (NIDDM), in 42 VAD subjects, in 23 VAD subjects with NIDDM, in 15 subjects with CRF and in 12 subjects with EH, we determined the PMN membrane fluidity, obtained marking unstimulated PMN cells with fluorescent probe 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), and considering the fluorescence polarization degree, and the PMN cytosolic Ca2+ content, obtained marking unstimulated PMN cells with the fluorescent probe Fura2-AM and considering the ratio between the Fura2-Ca2+ complex and the unchelated Fura 2 fluorescence intensity. From the obtained data it is evident that PMN membrane fluidity does not distinguish normals from IDDM subjects, NIDDM subjects, VAD subjects with and without NIDDM, CRF subjects and hypertensives. PMN cytosolic Ca2+ content, in comparison with normal controls, is significantly increased in VAD subjects (p < 0.01), in VAD subjects with NIDDM (p < 0.001), in CRF subjects (p < 0.001) and in hypertensives (p < 0.05). No correlation was found between PMN membrane fluidity and PMN cytosolic Ca2+ content. The study of these PMN parameters can be useful in the understanding of the role of leukocytes in the vascular damage that characterizes these clinical conditions.

Exercise test: trend of the leukocyte flow properties, polymorphonuclear membrane fluidity and cytosolic Ca2+ content in normals, in subjects with previous acute myocardial infarction and in subjects with aortocoronary by-pass.

We evaluated, during an exercise test, the leukocyte flow properties, the polymorphonuclear leukocyte (PMN) membrane fluidity and PMN cytosolic Ca2+ content in normals, in subjects with previous acute myocardial infarction (AMI) and in subjects previously submitted to a aortocoronary by-pass. Leukocyte flow properties were evaluated using the St. George filtrometer. Examination of the PMN membrane fluidity was effected employing the probe TMA-DPH; while evaluation of the PMN cytosolic Ca2+ content was carried out using the probe Fura 2-AM. At baseline, in both cardiopathic groups a significant difference in PMN filtration parameters and in PMN cytosolic Ca2+ content was evident compared to normals. In normals, at peak of exercise, there was an evident reduction of mononuclear filtration parameters, while during recovery a slight increase of the PMN cytosolic Ca2+ content was observed. In subjects with previous AMI and in subjects with aortocoronary by-pass, however, we observed, at peak of exercise, a decrease of the mononuclear filtration parameters, a reduction of the PMN membrane fluidity and an increase of the PMN cytosolic Ca2+ content. In both groups, the changes in PMN membrane fluidity and cytosolic Ca2+ content remained during recovery. The trend of the PMN membrane fluidity and cytosolic Ca2+ content found in the cardiopathic subjects during the exercise test suggest the PMN activation may be more evident in these subjects.

Leukocyte flow properties, polymorphonuclear membrane fluidity and cytosolic Ca2+ content in subjects with vascular atherosclerotic disease.

In a group of subjects with monovascular atherosclerotic disease (MVAD) and in a group of subjects with polyvascular atherosclerotic disease (PVAD) we evaluated white blood cell (WBC) filtration (unfractionated, mononuclear -MN-, polymorphonuclear -PMN- cells), using the St. George Filtrometer and considering respectively the initial relative flow rate (IRFR) and the clogging rate (CR), the polymorphonuclear leukocyte membrane fluidity, employing the fluorescent probe 1.4-(trimethylamino)-phenyl-4-phenylhexatriene (TMA-DPH) and calculating the fluorescence polarization degree, and the polymorphonuclear leukocyte cytosolic Ca2+ content, adopting the fluorescent probe Fura 2-AM. Only the filtration parameters (IRFR, CR) of unfractionated WBCs discriminate normals from MVAD and PVAD subjects, and also monovascular and polyvascular VAD subjects between them. The filtration parameters of mononuclear and polymorphonuclear leukocytes do not distinguish normals from MVAD and PVAD subjects. PMN membrane fluidity does not differentiate normals from MVAD and PVAD subjects, while PMN cytosolic Ca2+ content discriminates normals from MVAD and PVAD subjects, but does not distinguish the two groups of VAD subjects. In conclusion, in subjects with vascular atherosclerotic disease we noted an alteration of the unfractionated leukocyte flow properties, more evident in PVAD subjects, and an increase of the PMN cytosolic Ca2+ content.

Behavior of the polymorphonuclear leukocyte membrane fluidity and cystolic Ca2+ content in vascular atherosclerotic disease with and without non-insulin-dependent diabetes mellitus.

In 71 subjects with vascular atherosclerotic disease (VAD), in 32 VAD subjects with non-insulin-dependent diabetes mellitus (NIDDM) and in 31 normal controls, we evaluated polymorphonuclear leukocyte (PMN) membrane fluidity and PMN cytosolic Ca2+ content. The PMN membrane fluidity was obtained by marking intact and unstimulated PMN cells with fluorescent probe 1-[4-(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) and the PMN cytosolic Ca2+ content was obtained by marking intact and unstimulated PMN cells with the fluorescent probe Fura 2-AM. From the obtained results, it is evident that PMN membrane fluidity does not differentiate normals from VAD subjects and VAD subjects with NIDDM, and normals from subjects with monovascular disease (MVAD) and polyvascular disease (PVAD) with and without NIDDM. The PMN cytosolic Ca2+ content is significantly increased in VAD subjects and VAD subjects with NIDDM, and also in MVAD and PVAD subjects with and without NIDDM. A positive correlation is present between PMN membrane fluidity and PMN cytosolic Ca2+ content in normals and VAD subjects, but not in VAD subjects with NIDDM. In conclusion, in VAD subjects with and without NIDDM, an increase of the PMN cytosolic Ca2+ content is present; this increase might be related to the PMN spontaneous activation.

Cytosolic Ca2+ content and membrane fluidity of platelets and polymorphonuclear leucocytes in diabetes mellitus.

Considering the role played by platelets and leucocytes in diabetic disease and keeping in mind the strong correlation between functional and metabolic aspects that characterizes this clinical condition, we evaluated, in two groups of diabetics, respectively the platelet and polymorphonuclear (PMN) cytosolic Ca2+ content (employing the fluorescent probe Fura 2-AM) and membrane fluidity (using the fluorescent probe TMA-DPH and considering the fluorescence polarization degree, inversely related to the membrane fluidity). From the obtained results, it is evident that the platelet cytosolic Ca2+ content does not distinguish normals from diabetics of type 1 and 2; the platelet membrane fluidity instead does not discriminate normals from diabetics, but differentiates diabetics of type 1 and 2 (type 1 = 0.284 +/- 0.015; type 2 = 0.314 +/- 0.018; p < 0.001). PMN cytosolic Ca2+ content and membrane fluidity do not discriminate normals from diabetics. In the two groups of diabetics none of the platelet and PMN parameters (cytosolic Ca2+ content and membrane fluidity) are related to the glycometabolic pattern.

Membrane fluidity, membrane lipid pattern, and cytosolic Ca2+ content in platelets from a group of type II diabetic patients with macrovascular complications.

OBJECTIVE: To evaluate platelet membrane fluidity and some platelet metabolic parameters in type II diabetic patients with macrovascular complications. RESEARCH DESIGN AND METHODS: In a group of 21 type II diabetic patients with macrovascular complications, we evaluated platelet membrane fluidity [marking intact resting platelets with the fluorescent probe 1,4-(trimethylamino)-phenyl-4-phenylhexatriene (TMA-DPH)], platelet membrane lipid pattern (cholesterol:phospholipid [C:PL] ratio and individual phospholipids), and platelet cytosolic Ca2+ content (marking intact resting platelets with the fluorescent probe Fura 2AM). RESULTS: Platelet membrane fluidity is decreased in type II diabetic patients with macrovascular complications compared with normal subjects (P < 0.001). Platelet membrane C:PL ratio and cytosolic Ca2+ content do not discriminate normal subjects from diabetic patients, and for individual phospholipids, only phosphatidylethanolamine is decreased in diabetic patients compared with control subjects (P = 0.051). In normal subjects, the polarization degree of TMA-DPH is related to phosphatidylserine (P < 0.05) and phosphatidylcholine (P < 0.05), and in diabetic patients the polarization degree of TMA-DPH is related to C:PL ratio (P < 0.05) and sphyngomyelin (P < 0.05). CONCLUSIONS: In type II diabetic patients with macrovascular complications, we observed an abnormality of platelet membrane fluidity, which may contribute to platelet functional alteration present in this clinical condition.