[New possibilities in antibacterial treatment of intra-abdominal infections caused by multiresistant microflora]. / Novye vozmozhnosti antibakterial'noi terapii intraabdominal'nykh infektsii, vyzvannykh polirezistentnoi mikrobnoi floroi.

AIM: To determine the place of new drugs with activity against multidrug resistant strains of microorganisms in the treatment of complicated intraabdominal infections. MATERIAL AND METHODS: The incidence and distribution of pathogens isolated from intra-abdominal specimens in patients with intra-abdominal infections are analyzed. RESULTS: The current situation on the growth of resistant strains among pathogens causing intra-abdominal infections is rewied. New combined drugs for the treatment of multidrug resistant infections - ceftolozane/tazobactam and ceftazidim/avibactam plus metronidazole, has been suggested. Their potential role in empiric and targeted antibacterial treatment of complicated intraabdominal infections is defined. CONCLUSION: Taking into consideration local monitoring data and risk factors of multi resistant strains Ceftolozane/tazobactam in combination with metronidazole can be used in empiric regime of treatment. Due to the high activity on carbapenem resistant strains of Klebsiella pneumonia and the lack of alternatives, it is advisable to use Ceftazidim/avibactam for the targeted therapy.

Influence of external pH on two types of low-voltage-activated calcium currents in primary sensory neurons of rats.

The influence of extracellular pH (pH(o)) on low-voltage-activated calcium channels of acutely isolated DRG neurons of rats was examined using the whole cell patch-clamp technique. It has been found that in the neurons of middle size with capacitance C=60+/-4.8 pF (mean+/-S.E., n=8) extracellular acidification from pH(o) 7.35 to pH(o) 6.0 significantly and reversibly decreased LVA calcium current densities by 75+/-3.7%, shifted potential for half-maximal activation to more positive voltages by 18.7+/-0.6 mV with significant reduction of its voltage dependence. The half-maximal potential of steady-state inactivation shifted to more positive voltages by 12.1+/-1.7 mV (n=8) and also became less voltage dependent. Dose-response curves for the dependence of maximum values of LVA currents on external pH in neurons of middle size have midpoint pK(a)=6.6+/-0.02 and hill coefficient h=0.94+/-0.04 (n=5). In small cells with capacitance C=26+/-3.6 pF (n=5), acidosis decreased LVA calcium current densities only by 15.3+/-1.3% and shifted potential for half-maximal activation by 5.5+/-1.0 mV with reduction of its voltage dependence. Half-maximal potential of steady-state inactivation shifted to more positive voltages by 10+/-1.6 mV (n=4) and also became less voltage dependent. Dose-response curves for the dependence of maximum values of LVA currents on external pH in neurons of small size have midpoint pK(a)=7.9+/-0.04 and hill coefficient h=0.25+/-0.1 (n=4). These two identified types of LVA currents besides different pH sensitivity demonstrated different kinetic properties. The deactivation of LVA currents with weak pH sensitivity after switching off depolarization to -30 mV had substantially longer decay time than do currents with strong pH sensitivity (tau(d) approximately 5 ms vs. 2 ms respectively). It was found that the prolongation of depolarization steps slows the subsequent deactivation of T-type currents in small DRG neurons. Deactivation traces in these neurons were better described by the sum of two exponentials. Thus, we suppose that T-type channels in small DRG neurons are presented mostly by alpha1I subunit. We suggest that these two types of LVA calcium channels with different sensitivity to external pH can be differently involved in the origin of neuropathic changes.

Role of mitochondria in intracellular calcium signaling in primary and secondary sensory neurones of rats.

The participation of different calcium-regulated mechanisms in the generation of cytosolic Ca(2+) transients during neuronal excitation has been compared in isolated large and small primary (dorsal root ganglia (DRG)) and secondary (spinal dorsal horn (DH)) rat sensory neurones. As it was shown before in murine primary sensory neurones the application of mitochondrial protonophore CCCP by itself induced only small elevation of [Ca(2+)](i). However, its preceding application substantially increased the peak amplitude of depolarization-induced transients. Application of CCCP immediately after termination of the depolarizing pulse induced in both types of primary neurones a massive release of Ca(2+) from mitochondria into the cytosol. In secondary neurones application of CCCP by itself induced a substantial release of Ca(2+) from the mitochondria, but its preceding application resulted in only an insignificant increase in the peak amplitude of depolarization-triggered calcium transients. Application of CCCP immediately after termination of depolarization elicited a small release of Ca(2+), which became more pronounced when the application was delayed. Preceding application of CCCP increased the amplitude of the transients induced by caffeine-triggered Ca(2+) release from the endoplasmic reticulum in secondary neurones and did not affect those in large primary neurones. These findings may be explained by substantial differences in the density and distribution of mitochondria in the cytosol of primary and secondary sensory neurones. This suggestion was confirmed electronmicroscopically, showing a much lower density of mitochondria near plasmalemma in secondary sensory neurones and predominant clustered location of mitochondria beneath the plasmalemma in the primary cells. The possible functional importance of these differences is discussed.

Role of mitochondrial dysfunction in calcium signalling alterations in dorsal root ganglion neurons of mice with experimentally-induced diabetes.

The role of mitochondrial dysfunction in alterations of calcium signalling in primary sensory neurons has been studied in mice with streptozotocin-induced and genetically predisposed diabetes mellitus before and after additional treatment with insulin infusions. Cytosolic calcium transients triggered by membrane depolarization were measured using a membrane-permeable form of fluorescent indicator indo-1, and their changes after application of mitochondrial uncoupler carbonyl cyanide m-chlorphenylhydrazone were compared in cells of control and diabetic animals. Considerable prolongation of residual elevation of cytosolic calcium after termination of membrane depolarization was observed in diabetic mice, which was expressed mainly in small-sized (nociceptive) neurons. This correlated with the level of hyperglycemia, which was maximal in cells from streptozotocin-treated mice. Insulin partly reversed these changes. Carbonyl cyanide m-chlorophenylhydrazone application to neurons of control mice enlarged the peak of calcium transients and decreased residual calcium elevations, indicating that mitochondria in physiological conditions participate in shaping of these transients by diminishing their peak due to rapid uptake of calcium ions and by prolonging them due to subsequent slow calcium release back into the cytosol. Depression of the calcium accumulating function of mitochondria by carbonyl cyanide m-chlorophenylhydrazone eliminated these changes. The prolonged residual elevation of cytosolic calcium characteristic for neurons of diabetic animals was also eliminated by carbonyl cyanide m-chlorophenylhydrazone, confirming the suggestion that such elevation is determined mainly by mitochondrial dysfunction, the latter being dependent on the level of hyperglycemia. Predominant expression of such changes in small-sized neurons can be explained by the absence in them of effective calcium-buffering by the endoplasmic reticulum. Possible role of the described calcium signalling changes in the origin of neuropathic syndromes is discussed.

Effect of streptozotocin-induced diabetes on the activity of calcium channels in rat dorsal horn neurons.

We have previously found that spinal dorsal horn neurons from streptozotocin-diabetic rats, an animal model for diabetes mellitus, show the prominent changes in the mechanisms responsible for [Ca2+]i regulation. The present study aimed to further characterize the effects of streptozotocin-induced diabetes on neuronal calcium homeostasis. The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in Fura-2AM-loaded dorsal horn neurons from acutely isolated spinal cord slices using fluorescence technique. We studied Ca2+ entry through plasmalemmal Ca2+ channels during potassium (50 mM KCl)-induced depolarization. The K+-induced [Ca2+]i elevation was inhibited to a different extent by nickel ions, nifedipine and omega-conotoxin suggesting the co-expression of different subtypes of plasmalemmal voltage-gated Ca2+ channels. The suppression of [Ca2+]i transients by Ni2+ (50 microM) was the same in control and diabetic neurons. On the other hand, inhibition of [Ca2+]i transients by nifedipine (50 microM) and omega-conotoxin (1 microM) was much greater in diabetic neurons compared with normal animals. These data suggest that under diabetic conditions the activity of N- and L- but not T-type voltage-gated Ca2+ channels substantially increased in dorsal horn neurons.

Changes in calcium signalling in dorsal horn neurons in rats with streptozotocin-induced diabetes.

Intracellular calcium signalling was studied in the dorsal horn from neurons of rats with streptozotocin-induced diabetes versus control animals. The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in Fura-2 acetoxymethyl ester-loaded dorsal horn neurons from acutely isolated spinal cord slices using a fluorescence technique. The recovery of depolarization-induced [Ca2+]i increase was delayed in diabetic neurons compared with normal animals. In normal neurons, [Ca2+]i after the end of KCl depolarization recovered to the basal level monoexponentially with a time constant of 8.0+/-0.5 s (n = 23), while diabetic neurons showed two exponentials in the [Ca2+]i recovery. The time constants of these exponentials were 7.2+/-0.5 and 23.0+/-0.6 s (n = 19), respectively. The amplitude of calcium release from caffeine-sensitive endoplasmic reticulum calcium stores became significantly smaller in diabetic neurons. The amplitudes of [Ca2+]i transients evoked by 30 mM caffeine were 268+/-29 nM (n = 13) and 31+/-9 nM (n = 17) in control and diabetic neurons, respectively. We conclude that streptozotocin-induced diabetes is associated with prominent changes in the mechanisms responsible for [Ca2+]i regulation, which presumably include a slowdown of Ca2+ elimination from the cytoplasm by the endoplasmic reticulum.

Calcium signal prolongation in sensory neurones of mice with experimental diabetes.

Depolarization-induced Ca2+ transients were studied in dorsal root ganglion neurones of different size (large, 30-45 microns; small, 18-25 microns in diameter) from normal and diabetic mice. Whereas in large neurones no definite changes in the amplitude and time course of the transients were observed, in small neurones the decay of transient became substantially prolonged during streptozotocin-induced and spontaneously occurring diabetes. As small and large neurones differ substantially in their mechanisms of Ca2+ transient termination, we conclude that the prolongation of Ca2+ transients, probably induced by chronic hyperglycaemia, is specific only for small sensory neurones (transmitting mostly nociceptive signals) and may be a cause of the increased pain sensitivity often accompanying this disease.

Changes in mitochondrial Ca2+ homeostasis in primary sensory neurons of diabetic mice.

Changes in neuronal Ca2+ homeostasis were studied on freshly isolated dorsal root ganglion neurons of adult control mice and mice with streptozotocin (STZ)-induced diabetes. The cytoplasmic free Ca2+ concentration ([Ca2+]in) was measured using indo-1 based microfluorimetry. The participation of mitochondria in [Ca2+]in homeostasis was determined by investigation of changes which occurred after addition of mitochondrial protonophore (CCCP) to the extracellular solution. In control cells 10 microM CCCP applied before membrane depolarization induced an increase of the amplitude of depolarization-induced [Ca2+]in transients and disappearance of their delayed recovery, indicating the participation of mitochondria in fast uptake of Ca2+ ions from the cytosol during the peak of the transient and subsequent slow release them back during its decay. In diabetic animals the increase of the peak transient amplitude under the action of CCCP became diminished in small (nociceptive) neurons and the delayed elevation of [Ca2+]in disappeared in both large and small neurons. It is concluded that in diabetic conditions substantial changes occur in the Ca2+ homeostatic functions of mitochondria, manifested by decreased Ca2+ uptake in small neurons and depressed Ca2+ release into the cytosol in all types of neurons.

The endoplasmic reticulum and mitochondria as elements of the mechanism of intracellular signaling in the nerve cell.

Experimental data obtained in our laboratory from studies of intracellular signals arising within nerve cells during excitation are summarized. Measurements of transmembrane ion currents in conditions of fixed membrane potential and intracellular free Ca ion concentrations, using fluorescent probes, yielded the time and spatial characteristics of transient elevations in the Ca concentration (the "calcium signal") in various types of mouse and rat neurons. These studies showed that intracellular structures-the endoplasmic reticulum and mitochondria-had significant roles in forming these signals; these structures can take up Ca from the cytosol and liberate Ca into the cytosol; the contribution of these processes was extremely variable, depending on the internal organization of different functional types of neurons.

Alkalinization-induced changes in intracellular calcium in rat spinal cord neurons.

It is well-known that pH changes can influence a lot of cellular processes. In this work, we have specifically studied the influence of alkalinization, which can be developed in spinal cord neurons during hyperventilation (respiratory alkalosis) and chronic renal failure (metabolic alkalosis) on calcium homeostasis. Application of Tyrode solution with increased pH (pH = 8.8) to secondary sensory neurons isolated from rat spinal dorsal horn induced elevation of intracellular free calcium concentration in the cytosol ([Ca2+]i) if applied after membrane depolarization. Repetitive application of alkaline solution led to disappearance of such elevations. Depletion of endoplasmic reticulum (ER) calcium stores by 30 mM caffeine almost completely blocked the effect of elevated extracellular pH. If caffeine-induced [Ca2+]i transients were evoked during alkalinization, their amplitudes were decreased by 41%. Preapplication of 500 nM ionomycin resulted in disappearance of alkalinization-induced [Ca2+]i transients, whereas prolonged applications (for 20 min) of 200 nM thapsigargin, a blocker of Ca2+ ATPase of the endoplasmic reticulum, resulted in disappearance of the rapid phase of the [Ca2+]i transients induced by alkalinization. Preapplication of the mitochondrial protonophore CCCP (10 microM) also induced changes in the alkalinization-induced calcium response--it lost its peak and was transformed into an irregular wave terminating in several seconds. The data obtained indicate that alkalinization induces an increase of [Ca2+]i level in the investigated neurons via a combined action of both intracellular Ca2+-accumulating structures--the endoplasmic reticulum and mitochondria. This suggestion was supported by morphological data that both structures in these neurons are tightly connected and may interact during release of accumulated calcium ions.

Diabetes-induced changes in calcium homeostasis and the effects of calcium channel blockers in rat and mice nociceptive neurons.

AIMS/HYPOTHESIS: Distal neuropathy is the most common complication of diabetes mellitus, making it important to reveal the cellular mechanisms leading to its development, one of which might be the alteration in intracellular calcium homeostasis in primary and secondary nociceptive neurons. We aimed to investigate these possible changes. METHODS: Control and streptozotocin-treated diabetic rats and mice were used. Changes in intracellular free calcium concentrations ([Ca(2+)]i) were measured fluorometrically in primary nociceptive neurons from dorsal root ganglia and in secondary nociceptive neurons from substantia gelatinosa of spinal dorsal horn slices. RESULTS: Measurements of [Ca(2+)]i increases induced in dorsal root ganglion and dorsal horn neurons by membrane depolarization did not show any substantial difference in their peak amplitudes in control and diabetic animals. However, a definite prolongation of the decay phase of the transients was observed under diabetic conditions. Caffeine application to dorsal root ganglion and dorsal horn neurons induced a transient elevation of [Ca(2+)]i which was less prominent in cells from diabetic animals. Short-term application of a calcium channel blocker nifedipine showed a substantial amplification of its action in diabetic neurons. However, chronic administration of nimodipine induced a clear increase in the peak values of transients in dorsal root ganglion neurons of diabetic animals compared with those of untreated animals. CONCLUSION/INTERPRETATION: The described changes of calcium signalling in nociceptive neurons could be the reason for the development of distal polyneuropathy and its symptoms in the early stages of diabetes mellitus.

Intracellular calcium homeostasis changes induced in rat spinal cord neurons by extracellular acidification.

Changes in intracellular Ca2+ induced by extracellular acidification to pH = 6 were studied in isolated rat spinal dorsal horn neurons using indo-1 fluorescent technique. In all neurons such treatment induced a decrease of basal [Ca2+]i level by 20.8%, preceded in some of them by temporary increase. The changes were completely reversible. The depolarization-induced [Ca2+]i transients became strongly and also reversibly depressed. If tested after termination of acidification, they demonstrated substantial prolongation of their decay phase, reaching 310% at 120 sec after the application of depolarization. To analyze the mechanisms of such changes, mitochondrial protonophore CCCP has been applied between the end of acidification and the depolarizing pulse. This completely eliminated the described slowing of the transients' decay. To the contrary, application of caffeine to induce Ca2+ release from the endoplasmic reticulum did not show significant changes in the corresponding [Ca2+]i transients. A conclusion is made that in mammalian neurons extracellular acidification, apart from inhibiting voltage-operated Ca2+ channels, also substantially alters the Ca2+ exchange function of mitochondria responsible for rapid accumulation of ions and their delayed release back into the cytosol.

Diabetes-induced abnormalities in ER calcium mobilization in primary and secondary nociceptive neurons.

Development of diabetic sensory polyneuropathy is associated with alterations in intracellular calcium homeostasis in primary and secondary nociceptive neurons. We have shown previously that in a model of streptozotocin (STZ)-induced diabetes, the calcium signal is prolonged and calcium release from ryanodine-sensitive calcium stores down-regulated in neurons of the nociceptive system. The aim of the present study was a more detailed characterization of calcium homeostasis in primary (dorsal root ganglia, DRG) and secondary (dorsal horn, DH) nociceptive neurons in STZ-induced diabetes. Fluorescence video-imaging was used to measure free cytosolic [Ca2+] ([Ca2+]i) in lumbar nociceptive neurons of control and streptozotocin-diabetic rats. Resting [Ca2+]i rose progressively in these neurons with the duration of diabetes and calcium mobilization from the endoplasmic reticulum (ER) decreased during diabetes. The amplitude of calcium release from both ryanodine- and IP3-sensitive calcium stores induced by caffeine, ionomycin, ATP or glutamate was significantly (P<0.01) lower in DRG and DH neurons from 6-week STZ-diabetic rats. Diabetes-induced changes in the calcium homeostasis were similar in DRG and DH neurons indicating that they might be general for many types of neurons from the central and peripheral nervous systems.