Nelson G. Publicover

Thermal decomposition based synthesis of Ag-In-S/ZnS quantum dots and their chlorotoxin-modified micelles for brain tumor cell targeting

Cadmium-free silver-indium-sulfide (Ag-In-S or AIS) chalcopyrite quantum dots (QDs) as well as their core-shell structures (AIS/ZnS QDs) are being paid significant attention in biomedical applications because of their low toxicity and excellent optical properties. Here we report a simple and safe synthetic system to prepare high quality AIS and AIS/ZnS QDs using thermal decomposition. The synthetic system simply involves heating a mixture of silver acetate, indium acetate, and oleic acid in dodecanethiol at 170 °C to produce AIS QDs with a 13% quantum yield (QY). After ZnS shell growth, the produced AIS/ZnS QDs achieve a 41% QY. To facilitate phase transfer and bioconjugation of AIS/ZnS QDs for cellular imaging, these QDs were loaded into the core of PLGA-PEG (5k:5k) based micelles to form AIS/ZnS QD-micelles. Cellular imaging studies showed that chlorotoxin-conjugated QD-micelles can be specifically internalized into U-87 brain tumor cells. This work discloses that the scalable synthesis of AIS/ZnS QDs and the facile surface/interface chemistry for phase transfer and bioconjugation of these QDs may open an avenue for the produced QD-micelles to be applied to the detection of endogenous targets expressed on brain tumor cells, or more broadly to cell- or tissue-based diagnosis and therapy.

Induction and organization of Ca2+ waves by enteric neural reflexes.

The motility of the gastrointestinal tract consists of local, non-propulsive mixing (pendular or segmental) and propulsive (peristaltic) movements. It is generally considered that mixing movements are produced by intrinsic pacemakers which generate rhythmic contractions, and peristalsis by intrinsic excitatory and inhibitory neural reflex pathways,,, but the relationship between mixing and peristalsis is poorly understood. Peristalsis is compromised in mice lacking interstitial cells of Cajal, suggesting that these pacemaker cells may also be involved in neural reflexes. Here we show that mixing movements within longitudinal muscle result from spontaneously generated waves of elevated internal calcium concentration which originate from discrete locations (pacing sites), spread with anisotropic conduction velocities in all directions, and terminate by colliding with each other or with adjacent neurally suppressed regions. Excitatory neural reflexes control the spread of excitability by inducing new pacing sites and enhancing the overall frequency of pacing, whereas inhibitory reflexes suppress the ability of calcium waves to propagate. We provide evidence that the enteric nervous system organizes mixing movements to generate peristalsis, linking the neural regulation of pacemakers to both types of gut motility.

Spontaneous release of nitric oxide inhibits electrical, Ca2+ and mechanical transients in canine gastric smooth muscle.

1. In canine antrum, rhythmic electrical activity consists of a rapid upstroke phase followed by a plateau depolarization. In response to slow waves, cytosolic Ca2+ ([Ca2+]cyt) and tension increased. 2. Addition of sodium nitroprusside (SNP, 0.5 microM) decreased the amplitude of the plateau phase of slow waves without significant effects on the upstroke depolarization. SNP also inhibited changes in [Ca2+]cyt and tension associated with the plateau potential. SNP induced a negative chronotropic effect at concentrations above 0.1 microM. 3. Similar to the effects of SNP, illumination of muscles during slow waves with ultraviolet (UV) light caused premature repolarization. UV illumination is known to release NO in some tissues. 4. L‐NG‐monomethyl‐arginine (L‐NMMA, 300 microM), Methylene Blue (MB, 5 microM) and oxyhaemoglobin (oxy‐Hb, 5 microM) increased the force of contractions. In contrast, L‐arginine (L‐Arg, 300 microM) decreased contractile force and antagonized the effects of L‐NMMA. 5. During the upstroke phase, SNP caused a small reduction in [Ca2+]cyt and a large reduction in force, suggesting that SNP caused a decrease in Ca2+ sensitivity. 6. In muscles permeabilized by alpha‐toxin, cyclic GMP (100 microM) and UV illumination inhibited Ca(2+)‐induced contraction (at pCa 5.5). 7. These data suggest that NO or NO‐related compounds are spontaneously released in gastric muscles. These agents have two effects on excitation‐contraction coupling: (i) inhibition (directly and/or indirectly) of the voltage‐dependent Ca2+ channels that participate in the plateau phase of slow waves, and (ii) reduction in the Ca2+ sensitivity of the contractile element.

Propagation and Neural Regulation of Calcium Waves in Longitudinal and Circular Muscle Layers of Guinea Pig Small Intestine

BACKGROUND & AIMS The relative movements of longitudinal muscle (LM) and circular muscle (CM) and the role that nerves play in coordinating their activities has been a subject of controversy. We used fluorescent video imaging techniques to study the origin and propagation of excitability simultaneously in LM and CM of the small intestine. METHODS Opened segments of guinea pig ileum were loaded with the Ca(2+) indicator fluo-3. Mucosal reflexes were elicited by lightly depressing the mucosa with a sponge. RESULTS Spontaneous Ca(2+) waves occurred frequently in LM (1.2 s(-1)) and less frequently in CM (3.2 min(-1)). They originated from discrete pacing sites and propagated at rates 8-9 times faster parallel (LM, 87 mm/s; CM, 77 mm/s) compared with transverse to the long axis of muscle fibers. The presence of Ca(2+) waves in one muscle layer did not affect the origin, rate of conduction, or range of propagation in the other layer. The extent of propagation was limited by collisions with neighboring waves or recently excited regions. Simultaneous excitation of both muscle layers could be elicited by mucosal stimulation of either ascending or descending reflex pathways. Neural excitation resulted in an increase in the frequency of Ca(2+) waves and induction of new pacing sites without eliciting direct coupling between layers. CONCLUSIONS Localized, spontaneous Ca(2+) waves occur independently in both muscle layers, promoting mixing (pendular or segmental) movements, whereas activation of neural reflexes stimulates Ca(2+) waves synchronously in both layers, resulting in strong peristaltic or propulsive movements.

Block by 4‐aminopyridine of a Kv1.2 delayed rectifier K+ current expressed in Xenopus oocytes.

1. The blocking action of 4‐aminopyridine (4‐AP) on a delayed rectifier Kv1.2 K+ channel expressed in oocytes was investigated at room temperature (22 degrees C) and physiological temperature (34 degrees C) using the double‐electrode voltage clamp and patch clamp techniques. 2. At room temperature, 4‐AP (100 microM) inhibition occurred only after activation of current. The rate of onset of block was dependent upon the length of time current was activated by a depolarizing step. Similarly, removal of block required current activation. The degree of steady‐state block by 4‐AP was not reduced by increasingly more depolarized step potentials. The degree of steady‐state block also did not change over the duration of a 1 s step. 3. When channels were nearly fully inactivated, 4‐AP produced no additional block of a subsequent depolarizing step, suggesting that 4‐AP did not bind when channels were in the inactivated state. In single channel experiments, 4‐AP decreased the mean open time in a dose‐dependent manner but did not alter the single‐channel current amplitude. 4. At 34 degrees C the I‐V relationship and inactivation curve shifted to more negative potentials. Increasing the temperature to 34 degrees C did not alter the degree of block by 4‐AP, although the rate of onset of block was greatly enhanced. 5. Results suggest that 4‐AP binds to the open state of the Kv1.2 channel and is trapped when the channel closes. 4‐AP cannot bind when the channel is closed or inactivated prior to the addition of the drug. C‐type inactivation and 4‐AP binding to the channel are mutually exclusive. A model for the proposed mechanism of action of 4‐AP on the Kv1.2 channel is proposed based on experimental data.

Participation of Ca2(+)-activated K+ channels in electrical activity of canine gastric smooth muscle.

1. The hypothesis that Ca2(+)‐activated K+ channels participate in the repolarization of electrical slow waves was tested in isolated cells and intact muscles of the canine gastric antrum. 2. Freshly dispersed cells from the gastric antrum liberally express large conductance channels that were characterized as Ca2(+)‐activated K+ channels by several criteria. 3. Mean slope conductance of these channels in symmetrical 140 mM‐KCl solutions was 265 +/‐ 25 pS and reversal potential was 1.3 +/‐ 3.3 mV. The reversal potential was shifted when K+ was partially replaced with Na+ in a manner consistent with the Nernst equation for the K+ gradient. 4. Open probability was studied in excised patches in solutions containing 10(‐7)‐10(‐6) M‐Ca2+ with holding potentials ranging from ‐100 to +100 mV. Resulting activation curves were fitted by Boltzmann functions. 5. Increasing [Ca2+] from 10(‐7) to 10(‐6) M shifted the half‐maximal activation from +99 to 0 mV. These data suggest that Ca2(+)‐activated K+ channels may be activated in the voltage range and [Ca2+]i occurring during the plateau phase of the slow wave. 6. In intact muscles loaded with the photolabile Ca2+ chelator, nitr‐5, photo‐activated release of Ca2+ during the slow wave cycle produced changes consistent with activation of Ca2(+)‐dependent outward currents. 7. The data are consistent with the idea that Ca2+ build‐up during electrical slow waves shifts the activation voltage of Ca2(+)‐activated K+ channels into the range of the plateau potential. Activation of these channels yields outward current and repolarization. 8. Since the force of contractions depends on slow wave amplitude and duration, regulation of these channels may be important in controlling gastric motility.

Cyclic AMP-mediated regulation of excitation-contraction coupling in canine gastric smooth muscle

1. Agonists known to increase cyclic AMP levels in gastrointestinal smooth muscles were studied in isolated circular muscles of the canine antrum to investigate the mechanisms of the inhibitory effects of these agents. 2. Muscles were electrically active, generating typical slow wave activity. Cytosolic Ca2+ ([Ca2+]cyt; measured by Indo‐1 fluorescence) and tension increased in response to slow waves. 3. Stimulation by isoprenaline (via beta 2‐receptors) or forskolin, in the presence or absence of acetylcholine, inhibited the plateau phase and reduced phasic [Ca2+]cyt and contractile responses. 4. Vasoactive intestinal peptide (VIP) and calcitonin gene‐related peptide (CGRP), had similar effects to isoprenaline and forskolin. 5. Increases in the plateau phase of slow waves and the associated increases in [Ca2+]cyt and tension caused by direct activation of voltage‐dependent Ca2+ channels by Bay K 8644 (0.1 microM) were also reduced by forskolin. 6. Isoprenaline and forskolin induced negative chronotropic effects, but VIP increased frequency. 7. At a given level of [Ca2+]cyt, contractions were greater under control conditions than in the presence of isoprenaline, VIP and CGRP, suggesting that part of the inhibition produced by these agents may be due to decreased Ca2+ sensitivity of the contractile apparatus. 8. Experiments performed on alpha‐toxin‐permeabilized muscles confirmed that cyclic AMP‐dependent effects involve reduced Ca2+ sensitivity of the contractile apparatus. Addition of cyclic AMP (3‐300 microM) caused a reduction in Ca(2+)‐induced contraction at a constant level of Ca2+ (pCa 5.5). 9. These results suggest that increased cyclic AMP and probably subsequent activation of protein kinase A: (i) decrease [Ca2+]cyt and contraction by an inhibition of Ca2+ influx during slow waves, and (ii) decrease the sensitivity of the contractile apparatus to [Ca2+]cyt. The membrane effects might occur directly by inhibition of Ca2+ channels or indirectly by increasing the open probability of K+ channels which would tend to cause premature repolarization of slow waves.

Time‐dependent changes in Ca2+ sensitivity during phasic contraction of canine antral smooth muscle.

1. Relationships between cytosolic Ca2+ concentration ([Ca2+]cyt), myosin light chain (MLC) phosphorylation and muscle tension were examined in circular smooth muscle of canine gastric antrum. 2. Electrical slow waves induced a transient increase in [Ca2+]cyt and muscle tension. [Ca2+]cyt increased before the initiation of contraction and reached a maximum before the peak of the phasic contractions. Following the first Ca2+ transient, a second rise in [Ca2+]cyt was often observed. The second Ca2+ transient was of similar magnitude to the first, but only in some cases was this increase in [Ca2+]cyt associated with a second phase of contraction. Relaxation occurred more rapidly than the restoration of resting levels of [Ca2+]cyt. 3. Acetylcholine (ACh; 3 x 10(‐7) M) increased the amplitude of Ca2+ transients, caused MLC phosphorylation and increased the force of contraction. The decay of contraction and MLC dephosphorylation preceded that of [Ca2+]cyt. 4. Increasing external K+ (to 25‐40 mM) caused a sustained increase in [Ca2+]cyt, but little change in resting tension. This suggests that the Ca2+ sensitivity decreased as [Ca2+]cyt increased. Increasing K+ to 59.5 mM further increased the level of [Ca2+]cyt, induced MLC phosphorylation and caused a transient contraction. When normal levels of K+ were restored, the rates of MLC dephosphorylation and relaxation exceeded the rate of decay in [Ca2+]cyt. 5. Removal of external Ca2+ in depolarized muscles decreased [Ca2+]cyt below the resting level without affecting resting tension. Readmission of Ca2+ to depolarized muscles caused force to develop at [Ca2+]cyt levels below the original resting level, suggesting that Ca2+ sensitivity was increased when the resting level of [Ca2+]cyt was decreased. 6. The phosphatase inhibitor, calyculin‐A (10(‐6) M), induced tonic contraction and MLC phosphorylation without an increase in [Ca2+]cyt. During these contractures, electrical activity caused transient increases in [Ca2+]cyt and phasic contractions which were superimposed upon the Ca(2+)‐independent contracture. In the presence of calyculin‐A, relaxation occurred in two phases. The initial, rapid phase of relaxation was not significantly affected by calyculin‐A, but the slow phase was significantly decreased. 7. These results suggest that the relationship between [Ca2+]cyt, MLC phosphorylation and contraction changes as a function of [Ca2+]cyt in canine antral muscles. This may be due to a Ca(2+)‐and time‐dependent phosphatase that regulates the level of myosin phosphorylation.

Sensitization of the contractile system of canine colonic smooth muscle by agonists and phorbol ester.

1. Sensitization of the contractile system in response to combinations of excitatory agonists acetylcholine (ACh), methacholine, histamine and neurokinin A (NKA) was investigated in colonic circular smooth muscle of dog, NKA (1 nM) potentiated the contractile response to 1 microM ACh, but did not increase the fura‐2 fluorescence ratio (R340/380). Contraction in response to low concentrations of either methacholine or histamine was potentiated significantly by 0.1 microM 4‐phorbol 12,13‐dibutyrate (PDBu), suggesting that activation of protein kinase C can potentiate contraction at threshold concentrations of agonists. 2. Variability in the sensitivity of the contractile system to Ca2+ was demonstrated over a range of agonist concentrations. KCl, ACh, histamine and NKA each produced a concentration‐dependent increase in the amplitude of phasic contractions and R340/380. However, ACh, histamine and NKA each induced maximal increases in R340/380 at concentrations less than that needed to induce maximum force. 3. In depolarized muscles, NKA (50 nM) and PDBu (1 microM) each increased the magnitude of tonic contraction with no change or a decrease in both R340/380 and myosin light chain phosphorylation. In alpha‐toxin‐permeabilized fibres, 0.1 microM PDBu and 1 microM NKA shifted the Ca(2+)‐force response to the left. Ca(2+)‐induced contractions were also potentiated by 100 microM GTP‐gamma‐S or 1 microM NKA plus 10 microM GTP. Potentiation of contraction by NKA and GTP was antagonized by 10 microM GDP‐beta‐S. 4. The results suggest that endogenous agonists acting via G‐proteins sensitize the contractile element of colonic smooth muscle in part by activation of protein kinase C. In some cases, sensitization may be secondary to increased myosin phosphorylation (ACh), but in other cases it appears to be independent of increased myosin light chain phosphorylation (NKA and PDBu). Therefore regulatory mechanisms in addition to myosin phosphorylation contribute to the apparent sensitization of the contractile system to Ca2+.

Ca2+ regulation of the contractile apparatus in canine gastric smooth muscle.

1. The relationships between cytosolic Ca2+ ([Ca2+]cyt; expressed as a fluorescence ratio at 400 nm and 500 nm using Indo‐1) and contractile force was examined in strips of circular smooth muscles of canine gastric antrum. Rhythmic increases in [Ca2+]cyt were observed and contractions were biphasic. 2. In most muscles (70%), the amplitude of the second phase of the Ca2+ transient was less than or equal to the first phase of the Ca2+ transient, but the second phase of the contraction was much smaller than the first phase, suggesting a decrease in Ca2+ sensitivity during the second contractile phase. In 30% of muscles, the amplitude of the second phase of the Ca2+ transient was 2‐ to 3‐fold greater than the first phase. In these muscles, the second phase of contraction was 10‐fold greater than the first phase of contraction. Thus, a non‐linear relationship between [Ca2+]cyt and force greatly amplifies force development when [Ca2+]cyt exceeds a threshold level. 3. Acetylcholine (ACh, 0.3‐1 microM) increased the amplitudes of Ca2+ transients and basal [Ca2+]cyt between phasic contractions. The increase in basal [Ca2+]cyt did not cause tone to develop. ACh increased the amplitude of Ca2+ transients 2‐ to 3‐fold and this was associated with a 15 to 20‐fold increase in the force of phasic contractions. Pentagastrin (0.5 nM) and cholecystokinin octapeptide (CCK, 40 nM) had similar effects on Ca2+ transients and phasic contractions. 4. Bay K 8644 (0.1 microM) and TEA (5 mM) also increased the amplitudes of Ca2+ transients by 2‐ to 3‐fold and phasic contractions by 15‐ to 30‐fold. There was no significant difference observed between the [Ca2+]cyt‐force relationships in the presence of agonists (i.e. ACh, pentagastrin and CCK) or when [Ca2+]cyt was increased by Bay K 8644 or TEA. These data suggest that agonist‐dependent increases in Ca2+ sensitivity may not significantly regulate the [Ca2+]cyt‐force relationship in antral muscles. 5. D600 (5 microM), added during stimulation with ACh (0.3 M), decreased [Ca2+]cyt and force without affecting the [Ca2+]cyt‐force relationship. 6. Mechanisms exist for agonist‐mediated enhancement of the Ca(2+)‐force relationship. In alpha‐toxin‐permeabilized antrum, ACh (10 microM) with GTP (100 microM) or GTP gamma S (100 microM) increased the Ca(2+)‐induced contraction at clamped levels of Ca2+. Phorbol 12,13‐dibutyrate (PDBu, 10 microM) also increased the contractile force at a given level of Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)