Omega-3 polyunsaturated fatty acids preserve retinal function in type 2 diabetic mice.

OBJECTIVE: Diabetic retinopathy (DR) is associated with hyperglycemia-driven microvascular pathology and neuronal compromise in the retina. However, DR is also linked to dyslipidemia. As omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) are protective in proliferative retinopathy, we investigated the capacity of ω-3PUFAs to preserve retinal function in a mouse model of type 2 diabetes mellitus (T2DM). DESIGN: Male leptin-receptor-deficient (db/db) mice were maintained for 22 weeks (4 weeks-26 weeks of life) on calorically and compositionally matched diets, except for 2% enrichment in either ω-3 or ω-6PUFAs. Visual function was assessed at 9, 14 and 26 weeks by electroretinography. Retinal capillary and neuronal integrity, as well as glucose challenge responses, were assessed on each diet. RESULTS: The ω-3PUFA diet significantly preserved retinal function in the mouse model of T2DM to levels similar to those observed in nondiabetic control mice on normal chow. Conversely, retinal function gradually deteriorated in db/db mice on a ω-6PUFA-rich diet. There was also an enhanced ability of ω-3PUFA-fed mice to respond to glucose challenge. The protection of visual function appeared to be independent of cytoprotective or anti-inflammatory effects of ω-3PUFAs. CONCLUSION: This study identifies beneficial effects of dietary ω-3PUFAs on visual function in T2DM. The data are consistent with dyslipidemia negatively impacting retinal function. As ω-3PUFA lipid dietary interventions are readily available, safe and inexpensive, increasing ω-3PUFA intake in diabetic patients may slow the progression of vision loss in T2DM.

Properties of the human muscle nicotinic receptor, and of the slow-channel myasthenic syndrome mutant epsilonL221F, inferred from maximum likelihood fits.

The mechanisms that underlie activation of nicotinic receptors are investigated using human recombinant receptors, both wild type and receptors that contain the slow channel myasthenic syndrome mutation, epsilonL221F. The method uses the program HJCFIT, which fits the rate constants in a specified mechanism directly to a sequence of observed open and shut times by maximising the likelihood of the sequence with exact correction for missed events. A mechanism with two different binding sites was used. The rate constants that apply to the diliganded receptor (opening, shutting and total dissociation rates) were estimated robustly, being insensitive to the exact assumptions made during fitting, as expected from simulation studies. They are sufficient to predict the main physiological properties of the receptors. The epsilonL221F mutation causes an approximately 4-fold reduction in dissociation rate from diliganded receptors, and a smaller increase in opening rate and mean open time. These are sufficient to explain the approximately 6-fold slowing of decay of miniature synaptic currents seen in patients. The distinction between the two binding sites was less robust, the estimates of rate constants being dependent to some extent on assumptions, e.g. whether an extra short-lived shut state was included or whether the EC50 was constrained. The results suggest that the two binding sites differ by roughly 10-fold in the affinity of the shut receptor for ACh in the wild type, and that in the epsilonL221F mutation the lower affinity is increased so the sites become more similar.

The quality of maximum likelihood estimates of ion channel rate constants.

Properties of maximum likelihood estimators of rate constants for channel mechanisms are investigated, to see what can and cannot be inferred from experimental results. The implementation of the HJCFIT method is described; it maximises the likelihood of an entire sequence of apparent open and shut times, with the rate constants in a specified reaction mechanism as free parameters. The exact method for missed brief events is used. Several methods for testing the quality of the fit are described. The distributions of rate constants, and correlations between them, are investigated by doing sets of 1000 fits to simulated experiments. In a standard nicotinic receptor mechanism, all nine free rate constants can be estimated even from one single channel recording, as long as the two binding sites are independent, even when the number of channels in the patch is not known. The estimates of rate constants that apply to diliganded channels are robust; good estimates can be obtained even with erroneous assumptions (e.g. about the value of a fixed rate constant or the independence of sites). Rate constants that require distinction between the two sites are less robust, and require that an EC50 be specified, or that records at two concentrations be fitted simultaneously. Despite the complexity of the problem, it appears that there exist two solutions with very similar likelihoods, as in the simplest case. The hazards that result from this, and from the strong positive correlation between estimates of opening and shutting rates, are discussed.

Effects of hypoxia and dithionite on catecholamine release from isolated type I cells of the rat carotid body.

1. Amperometric recordings were conducted to investigate the ability of hypoxia and anoxia to evoke quantal catecholamine secretion from isolated type I cells of the rat carotid body. 2. Hypoxia (PO2 8-14 mmHg) consistently failed to evoke catecholamine secretion from type I cells, when cells were perfused either at room temperature (21-24 C) or at 35-37 C, and regardless of whether Hepes- or HCO3-/CO2-buffered solutions were used. 3. Elevating extracellular [K+] caused concentration-dependent secretion from individual type I cells, with a threshold concentration of approximately 25 mM. In the presence of this level of extracellular K+, hypoxia (PO2 8-14 mmHg) caused a marked enhancement of secretion which was fully blocked by 200 microM Cd2+, a non-specific blocker of voltage-gated Ca2+ channels. 4. Anoxia (N2-equilibrated solution containing 0.5 mM dithionite) evoked exocytosis from type I cells when extracellular [K+] was 5 mM. This secretion was completely inhibited by removal of extracellular Ca2+, but was not significantly affected by Cd2+ (200 microM), Ni2+ (2 mM), Zn2+ (1 mM) or nifedipine (2 microM). Secretion was also observed when 0.5 mM dithionite was added to air-equilibrated solutions. 5. Anoxia also evoked secretion from chemoreceptive phaeochromocytoma (PC12) cells, which was wholly Ca2+ dependent, but unaffected by Cd2+ (200 microM). 6. Our results suggest that hypoxia can evoke catecholamine secretion from isolated type I cells, but only in the presence of elevated extracellular [K+]. This may be due to the cells being relatively hyperpolarized following dissociation. In addition, we have shown that dithionite evokes catecholamine release regardless of PO2 levels, and this release is due mainly to an artefactual Ca2+ influx pathway activated in the presence of dithionite.

Arachidonic acid inhibits both K+ and Ca2+ currents in isolated type I cells of the rat carotid body.

Whole-cell patch-clamp recordings were used to investigate the effects of arachidonic acid (AA) on K+ and Ca2+ channels in isolated rat type I carotid body cells. AA (2-20 microM) produced a concentration-dependent inhibition of both K+ currents and Ca2+ channel currents. The effects of AA on K+ currents were unaffected by indomethacin (5 microM), phenidone (5 microM) or 1-aminobenzotriazole (3 mM), suggesting that AA did not exert its effects via cyclo-oxygenase, lipoxygenase or cytochrome P-450 (cP-450) metabolism. Our results suggest that AA directly and non-selectively inhibits ionic currents in rat type I carotid body cells.

Developmental changes in isolated rat type I carotid body cell K+ currents and their modulation by hypoxia.

1. Whole-cell patch-clamp recordings were used to investigate possible age-related changes in K+ currents of type 1 carotid body cells isolated from the rat. K+ current density increased with age, as measured in cells isolated from 4-day-old, 10-day-old and adult rats (> or = 5 weeks old). 2. The proportion of current reversibly inhibited by high [Mg2+] (6 mM), low [Ca2+] (0.1 mM) solutions, indicative of the proportion of current attributable to activation of Ca(2+) -sensitive K+ (KCa) channels, was significantly smaller in cells of 4-day-old rats compared with 10-day-old rats, despite inward Ca2+ current densities being similar in these two age groups. Inhibition of K+ currents by high [Mg2+], low [Ca2+] solutions was similar in 10-day-old and adult type 1 cells. 3. Hypoxia (PO2, 16-23 mmHg) caused reversible reductions in type I cells from rats of all age groups. However, reductions seen in cells of 4-day-old rats were significantly smaller than those seen in cells of 10-day-olds and adults. The degree of hypoxic inhibition in these latter two groups was not significantly different. 4. In the presence of high [Mg2+], low [Ca2+] solutions, hypoxia (PO2, 16-23 mmHg) was without significant effect on residual K+ currents in cells from all age groups. 5. These observations indicate that K+ current density increases with postnatal age in the rat. Between days 4 and 10, there appears to be a predominant enhancement of KCa channels, and over the same age range hypoxic sensitivity of K+ currents increases. Our findings demonstrate that this latter observation arises because hypoxia selectively inhibits KCa channels in cells at all ages studied. These results suggest an important role for KCa channels in postnatal maturation of hypoxic chemoreception in the rat carotid body.

Electrochemical detection of K(+)-evoked quantal secretory events from isolated rat type I carotid body cells.

Using amperometric techniques, electrochemical events associated with vesicular transmitter release were recorded from isolated rat type I carotid body cells when exposed to a solution containing 50 mM K+. Events were enhanced in amplitude by preloading cells with the catecholamine precursor, L-beta-3,4-dihydroxyphenylalanine (L-DOPA). K(+)-evoked secretion was abolished by the non-selective Ca2+ channel blocker Cd2+ (100 microM) and markedly reduced by the L-type Ca2+ channel blocker nifedipine (5 microM). Our results indicate that secretion from isolated rat type I cells can be monitored electrochemically and we demonstrate a major role for L-type Ca2+ channels in mediating K(+)-evoked secretion.

Multiple effects of nordihydroguaiaretic acid on ionic currents in rat isolated type I carotid body cells.

1. The effects of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) on the ionic currents of rat carotid body type I cells were investigated by use of whole-cell and outside-out patch clamp techniques. 2. NDGA (5-50 microM) produced a concentration-dependent inhibition of whole-cell K+ currents at all activating test potentials (holding potential -70 mV). The time-course of the inhibition was also concentration-dependent and the effects of NDGA were only reversible following brief periods of exposure (<2 min). Another lipoxygenase inhibitor, phenidone (5 microM), was without effect on whole-cell K+ currents in carotid body type I cells. 3. NDGA (5-50 microM) also inhibited whole-cell Ca2+ channel currents (recorded with Ba2+ as charge carrier) in a concentration-dependent manner. 4. Isolation of voltage-gated K+ channels by use of high [Mg2+] (6 mM), low [Ca2+] (0.1 mM) solutions revealed a direct inhibition of the voltage-sensitive component of the whole-cell K+ current by NDGA (50 microM). 5. In excised, outside-out patches NDGA (20-50 microM) increased large conductance, Ca2+ activated K+ channel activity approximately 10 fold, an effect which could be reversed by either tetraethylammonium (10 mM) or charybdotoxin (30 nM). 6. It is concluded that NDGA activates maxi-K+ channels in carotid body type I cells and over the same concentration range inhibits voltage-sensitive K+ and Ca2+ channels. The inhibition of whole cell K+ currents seen is most likely due to a combination of direct inhibition of the voltage-sensitive K+ current and indirect inhibition of maxi-K+ channel activity through blockade of Ca2+ channels.

Hypoxic inhibition of K+ currents in isolated rat type I carotid body cells: evidence against the involvement of cyclic nucleotides.

Whole-cell patch-clamp recordings were used to evaluate the effects of the cyclic nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) on ionic currents in type I carotid body cells isolated from rat pups, and to investigate whether cyclic nucleotides are involved in K+ current inhibition by hypoxia. In the presence of 500 microM isobutylmethylxanthine, currents were not significantly modified by 8-bromo-cAMP (2 mM), dibutyryl-cAMP (5 mM) or 8-bromo-cGMP (2 mM). Currents were also unaffected by the phosphodiesterase (PDE)-resistant protein kinase A activators Sp-cyclic adenosine-3', 5'-monophosphorothioate (Sp-cAMPS) and Sp-8-bromoadenosine-3', 5'-monophosphorothioate (Sp-8-bromo-cAMPS) (50 microM), or by beta-phenyl-1,N2-ethenoguanosine-3',5'-cyclic monophosphate (PET-cGMP) (100 microM) or the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP; 500 microM). Ca2+ channel currents were also unaffected by Sp-8-Br-cAMPS, PET-cGMP and SNAP at the same concentrations. In the absence of cyclic nucleotide analogues, hypoxia (PO2 17-23 mmHg) reversibly inhibited K+ currents. This degree of hypoxic inhibition was not significantly altered by the PDE-resistant protein kinase A inhibitors Rp-cyclic adenosine-3', 5'-monophosphorothioate (Rp-cAMPS) (50 microM) or Rp-8-bromoadenosine-3',5'-monophosphorothioate (Rp-8-bromo-cAMPS) (200 microM). Similarly, PET-cGMP (100 microM) and SNAP (500 microM) did not alter the degree of inhibition caused by hypoxia. At the same concentrations used in type I cell experiments, Sp-8-bromo-cAMPS, PET-cGMP and SNAP completely relaxed isolated guinea-pig basilar arteries preconstricted with 20 mM K+-containing solutions. Our results indicate that cyclic nucleotides alone are not an important factor in the regulation by O2 tension of K+ currents in rat type I carotid body cells.

Ca2+ channel currents in type I carotid body cells of normoxic and chronically hypoxic neonatal rats.

Whole-cell patch-clamp recordings were used to study voltage-gated Ca2+ channel currents in type I carotid body cells of young rats born and reared in normoxia or in a chronically hypoxic (CH) environment (10% O2). Currents activated at potentials of -40 mV and more positive, and typically peaked at 0 mV in both groups of cells. Steady-state inactivation curves were similar in the two populations. Ca2+ currents were significantly larger in CH type I cells, but this was accounted for by the increased size of CH cells: current density was similar in both cell types. Nifedipine (5 microM) always partially inhibited currents and Bay K 8644 (2-5 microM) always enhanced currents, indicating the presence of L-type channels. In a small number of cells from each group, the N-type channel blocker omega-conotoxin GVIA caused partial, irreversible inhibition, but in most cells was without discernible effect. These results indicate that type I cells possess L-type Ca2+ channels, that N-type are expressed in some cells and that non-L, non-N-type channels are also present. Furthermore, chronic hypoxia does not appear to cause specific adaptive changes in the properties of Ca2+ channels in type I cells.

Effects of cytochrome P-450 inhibitors on ionic currents in isolated rat type I carotid body cells.

Hypoxic chemoreception in the carotid body involves selective inhibition of K+ channels in type I cells. We have investigated whether cytochrome P-450 may act as an O2 sensor coupling hypoxia to K+ channel inhibition, by investigating the actions of P-450 inhibitors to modulate channel activity (recorded using patch-clamp techniques) in type I cells isolated from 8-to 12-day-old rat pups. The imidazole antimycotic P-450 inhibitors miconazole and clotrimazole (1-10 microM) inhibited the Ca(2+)-activated (KCa) and voltage-gated K+ (Kv) currents in isolated type I cells. Single-channel recordings indicated that the KCa channels could be inhibited directly by miconazole. Miconazole also irreversibly inhibited Ca2+ channel currents. By contrast, acute application of the suicide substrate P-450 inhibitor, 1-aminobenzotriazole (1-ABT; 3 mM) was without effect on K+ or Ca2+ currents. Hypoxia (16-23 mmHg) reversibly inhibited K+ currents and prevented the inhibitory actions of miconazole. Furthermore, the inhibitory actions of miconazole could be partially reversed by hypoxia. Pretreatment of cells for 60 min with 3 mM 1-ABT substantially reduced the inhibitory actions of hypoxia on K+ currents. Our results indicate that imidazole antimycotic P-450 inhibitors can directly and nonselectively inhibit ionic channels in type I cells but, more importantly, provide evidence to suggest that hypoxic inhibition of K+ currents in type I cells is mediated in part at least by cytochrome P-450.

Imidazo[4,5-f]quinolines. 4. Synthesis and anthelmintic activity of a series of imidazo[4,5-f]quinolin-9-ols.

A series of 2-arylimidazo[4,5-f]quinolin-9-ols has been prepared by a multistep procedure from various 5-aminobenzimidazoles. These compounds possess a significant degree of anthelmintic activity against the mouse tapeworm Hymenolepis nana. The most active compound is the 2-(2-furyl) analogue. Additional anthelmintic testing is reported for this compound.

Anthelmintic 1-cinnamamido-2,4-imidazolidinediones.

A series of 1-(substituted cinnamamido)-2,4-imidazolidinediones has been prepared from the corresponding cinnamoyl chlorides and 1-amino-2,4-imidazolidinedione hydrochloride in pyridine. These compounds possess a significant degree of anthelmintic activity against the mouse pinworm Syphacia obvelata. The most active compounds are those substituted with halogen or cyano groups.